Anti-IGF-IR and/or anti-insulin/IGF-I hybrid receptors antibodies and uses thereof

ABSTRACT

The present invention relates to methods of identifying IGF-IR modulators and hybrid-R modulators comprising contacting IGF-IR with a humanized anti-IGF-IR antibody and contacting hybrid-R with a humanized anti-hybrid-R antibody, respectively.

CROSS-REFERENCE TO PRIORITY/PCT/PROVISIONAL APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/388,534, filed on Feb. 19, 2009, now U.S. Pat. No.8,101,180, which is a divisional application of U.S. patent applicationSer. No. 11/012,353, filed on Dec. 16, 2004, now U.S. Pat. No.7,553,485, issued Jun. 30, 2009, which is a continuation-in-part of U.S.patent application Ser. No. 10/735,916, filed on Dec. 16, 2003, now U.S.Pat. No. 7,241,444, issued Jul. 10, 2007, which is acontinuation-in-part of PCT/FR 03/00178 filed in France on Jan. 20,2003, which claims priority from FR 0200653 filed in France on Jan. 18,2002, FR 0200654 filed in France on Jan. 18, 2002, FR 0205753 filed inFrance on May 7, 2002, and FR 0308538 filed in France on Jul. 11, 2003,the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION Technical Field of the Invention

The present invention relates to novel antibodies capable of bindingspecifically to the human insulin-like growth factor I receptor(hereinafter referred as “IGF-IR”) and/or the Insulin/IGF-I hybridreceptor, isoform(s) A and/or B, (hereinafter referred as “hybrid-R” orsometimes as “hybrid-Rs”, “hybrid-RsA” or “hybrid-RsB”) and/or capableof specifically inhibiting the tyrosine kinase activity of said IGF-IRand/or hybrid-R, especially monoclonal antibodies of murine, chimericand humanized origin, as well as the amino acid and nucleic acidsequences coding for these antibodies. The invention likewise comprisesthe use of these antibodies as a medicament for the prophylactic and/ortherapeutic treatment of cancers overexpressing, or with an abnormalactivation of, IGF-IR and/or hybrid-R or any pathology connected withthe overexpression, or an abnormal activation, of said receptor(s) aswell as in processes or kits for diagnosis of illnesses connected withthe overexpression, or an abnormal activation, of the IGF-IR and/orhybrid-R. The invention finally comprises products and/or compositionscomprising such antibodies in combination with anti-EGFR antibodiesand/or compounds and/or anti-cancer agents or agents conjugated withtoxins and their use for the prevention and/or the treatment of certaincancers.

The insulin-like growth factor I receptor called IGF-IR is a receptorwith tyrosine kinase activity having 70% homology with the insulinreceptor (hereinafter referred as “IR”). Sequence(s) coding for IGF-IRare registered under Accession Number NM_(—)000875 in the NCBI Genbank.Another data, without limitation and incorporated herein by reference,describing IGF-IR sequence(s) is Ulrich et al., 1986, EMBO J.,5(10):2503-2512.IGF-IR is a glycoprotein of molecular weightapproximately 350,000.It is a hetero-tetrameric receptor of which eachhalf-linked by disulfide bridges—is composed of an extracellularα-subunit and of a transmembrane β-subunit (see FIG. 1). IGF-IR bindsits native ligands, preferably IGF1 and IGF2, with a very high affinity(Kd #1 nM) but is equally capable of binding to insulin with an affinity100 to 1000 times less. Conversely, the IR binds insulin with a veryhigh affinity although the IGFs only bind to the IR with a 100 timeslower affinity. The tyrosine kinase domain of IGF-IR and of IR has avery high sequence homology although the zones of weaker homologyrespectively concern the cysteine-rich region situated on the α-subunitand the C-terminal part of the β-subunit. The sequence differencesobserved in the α-subunit are situated in the binding zone of theligands and are therefore at the origin of the relative affinities ofIGF-IR and of IR for the IGFs and insulin respectively. The differencesin the C-terminal part of the β-subunit result in a divergence in thesignaling pathways of the two receptors; IGF-IR mediating mitogenic,differentiation and antiapoptosis effects, while the activation of theIR principally involves effects at the level of the metabolic pathways(Baserga et al., Biochim. Biophys. Acta, 1332:F105-126, 1997; BasergaR., Exp. Cell. Res., 253:1-6, 1999).

The cytoplasmic tyrosine kinase proteins are activated by the binding ofthe ligand to the extracellular domain of the receptor. The activationof the kinases in its turn involves the stimulation of differentintra-cellular substrates, including IRS-1, IRS-2, Shc and Grb 10(Peruzzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999). Thetwo major substrates of IGF-IR are IRS and Shc which mediate, by theactivation of numerous effectors downstream, the majority of the growthand differentiation effects connected with the attachment of the IGFs tothis receptor (FIG. 2). The availability of substrates can consequentlydictate the final biological effect connected with the activation of theIGF-IR. When IRS-1 predominates, the cells tend to proliferate and totransform. When Shc dominates, the cells tend to differentiate(Valentinis B. et al., J. Biol. Chem. 274:12423-12430, 1999). It seemsthat the route principally involved for the effects of protectionagainst apoptosis is the phosphatidyl-inositol 3-kinases (PI 3-kinases)route (Prisco M. et al., Horm. Metab. Res., 31:80-89, 1999; Peruzzi F.et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999).

The role of the IGF system in carcinogenesis has become the subject ofintensive research in the last ten years. This interest followed thediscovery of the fact that in addition to its mitogenic andantiapoptosis properties, IGF-IR seems to be required for theestablishment and the maintenance of a transformed phenotype. In fact,it has been well established that an overexpression or a constitutiveactivation of IGF-IR leads, in a great variety of cells, to a growth ofthe cells independent of the support in media devoid of fetal calfserum, and to the formation of tumors in nude mice. This in itself isnot a unique property since a great variety of products of overexpressedgenes can transform cells, including a good number of receptors ofgrowth factors. However, the crucial discovery which has clearlydemonstrated the major role played by IGF-IR in the transformation hasbeen the demonstration that the R-cells, in which the gene coding forIGF-IR has been inactivated, are totally refractory to transformation bydifferent agents which are usually capable of transforming the cells,such as the E5 protein of bovine papilloma virus, an overexpression ofEGFR or of PDGFR, the T antigen of SV 40, activated ras or thecombination of these two last factors (Sell C. et al., Proc. Natl. Acad.Sci., USA, 90: 11217-11221, 1993; Sell C. et al., Mol. Cell. Biol.,14:3604-3612, 1994; Morrione A. J., Virol., 69:5300-5303, 1995; CoppolaD. et al., Mol. Cell. Biol., 14:4588-4595, 1994; DeAngelis T et al., J.Cell. Physiol., 164:214-221, 1995).

IGF-IR is expressed in a great variety of tumors and of tumor lines andthe IGFs amplify the tumor growth via their attachment to IGF-IR. Otherarguments in favor of the role of IGF-IR in carcinogenesis come fromstudies using murine monoclonal antibodies directed against the receptoror using negative dominants of IGF-IR. In effect, murine monoclonalantibodies directed against IGF-IR inhibit the proliferation of numerouscell lines in culture and the growth of tumor cells in vivo (Arteaga C.et al., Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys.Res. Com., 196:92-98, 1993; Zia F et al., J. Cell. Biol., 24:269-275,1996; Scotlandi K et al., Cancer Res., 58:4127-4131, 1998). It haslikewise been shown in the works of Jiang et al., (Oncogene,18:6071-6077, 1999) that a negative dominant of IGF-IR is capable ofinhibiting tumor proliferation.

For the first time, data illustrating the recognition of IGF-IR and/orhybrid-R by the same monoclonal antibody able to inhibit specifically,in vitro and in vivo, the tumoral growth, thus allowing to treat cancer,more particularly breast cancer, able to conjointly express the tworeceptor types are shown in the present example (see particularlyexample 26). Actually, the capacity of 7C10 and h7C10 to recognizeand/or inhibit the tyrosine kinase activity of IGF-IR and/or hybrid-Rallow to avoid the escape of tumor consequent upon the expression, orthe abnormal activation, of this hybrid-R. Such an antibody could be aninnovative therapeutic compound of a essential interest for thetreatment of cancer.

Cancer pathologies are characterized by an uncontrolled cellular growth.In several cancer, growth factors are specifically binding with theirreceptors and then transmit growth, transformation and/or survivalsignals to the tumoral cell. The growth factor receptors over-expressionat the tumoral cell surface is largely described (Salomon D S et al.,Crit. Rev. Oncol. Hematol. 1995, 19:183; Burrow S. et al., J. Surg.Oncol., 1998, 69:21; Hakam A. et al., Hum. Pathol, 1999, 30:1128; RailoM. J. et al., Eur. J. Cancer, 1994, 30:307; Happerfield L. C. et al., J.Pathol., 1997, 183:412). This over-expression, or abnormal activation,leading to a direct perturbation of cellular growth regulationmechanisms, can also affect the cell sensibility to induced apoptose byclassical chemotherapies or radiotherapies.

During last few years, it has been show that the targeting of growthfactor receptors, like EGFR or Her2/neu over-expressed on the tumoralcell surface, with respectively humanized (herceptin®) or chimeric(C225) antibodies results in an significant inhibition of the tumoralgrowth on patients and in a significant increase of the efficacity ofclassical chemotherapy treatments (Carter P., Nature Rev. Cancer, 2001,1(2):118; Hortobagyi G. N., Semin. Oncol., 2001, 28:43; Herbst R. S. etal., Semin. Oncol., 2002, 29:27). Other receptors like IGF-IR or VEGF-R(for vascular endothelial growth factor receptor) have been identifiedas potential target in several preclinical studies.

More particularly, IGF-IR is part of the tyrosine kinase receptors. Itshows a high homology with the Insulin receptor (IR) which exist undertwo isoforms A and B.

Sequences of IR, isoforms A and B, are registered under AccessionNumbers X02160 and M10051, respectively, in the NCBI Genbank. Otherdatas, without limitations, relating to IR are incorporated herein byreferences (Vinten et al., 1991, Proc. Natl. Acad. Sci., USA,88:249-252; Belfiore et al., 2002, The Journal of Biological Chemistry,277:39684-39695; Dumesic et al., 2004, The Journal of Endocrinology &Metabolism, 89(7):3561-3566).

The IGF-IR and IR are tetrameric glycoproteins composed of twoextracellular α- and two transmembrane β-subunits linked by disulfidebonds. Each α-subunit, containing the ligand-binding site isapproximately 130- to 135-kDa, whereas each β-subunit containing thetyrosine kinase domain is approximately 90- to 95-kDa. These receptorsshare more than 50% overall amino acid sequence similarity and 84%similarity in the tyrosine kinase domain. After ligand binding,phosphorylated receptors recruit and phosphorylate docking proteins,including the insulin receptor substrate-1 protein family (IRS1), Gab1and Shc (Avruch, 1998, Mol. Cell. Biochem., 182, 31-48; Roth et al.,1988, Cold Spring Harbor Symp. Quant. Biol. 53:537-543; White, 1998,Mol. Cell. Biochem., 182:3-11; Laviola et al., 1997, J. Clin. Invest.,99:830-837; Cheatham et al., 1995, Endocr. Rev. 16:117-142), leading tothe activation of different intracellular mediators. Although both theIR and IGF-IR similarly activate major signaling pathways, differencesexist in the recruitment of certain docking proteins and intracellularmediators between both receptors (Sasaoka et al., 1996, Endocrinology137:4427-4434; Nakae et al., 2001, Endocr. Rev. 22:818-835; Dupont andLe Roith, 2001, Horm. Res. 55, Suppl. 2:22-26; Koval et al., 1998,Biochem. J. 330:923-932). These differences are the basis for thepredominant metabolic effects elicited by IR activation and thepredominant mitogenic, transforming and anti-apoptotic effects elicitedby IGF-IR activation (De Meyts et al., 1995, Ann. N.Y. Acad. Sci.,766:388-401; Singh et al., 2000; Prisco et al., 1999, Horm. Metab. Res.31:80-89; Kido et al., 2001, J. Clin. Endocrinol. Metab. 86:972-979).Insulin binds with high affinity to the IR (100-fold higher than to theIGF-IR), whereas insulin-like growth factors (IGF1 and IGF2) bind to theIGF-IR with 100-fold higher affinity than to the IR.

The human IR exists in two isoforms, IR-A and IR-B, generated byalternative splicing of the IR gene that either excludes or includes 12amino acid residues encoded by a small exon (exon 11) at thecarboxy-terminus of the IR α-subunit. The relative abundance of IRisoforms is regulated by tissue specific and unknown factors (Moller etal., 1989, Mol. Endocrinol., 3:1263-1269; Mosthaf et al., 1990, EMBO J.,9:2409-2413). IR-B is the predominant IR isoform in normal adult tissues(adipose tissue, liver and muscle) that are major target tissues for themetabolic effects of insulin (Moller et al., 1989; Mosthaf et al.,1990). IR-A is the predominant isoform in fetal tissues and mediatesfetal growth in response to IGF2 (Frasca et al., 1999, Mol. Cell. Biol.,19:3278-3288), as also suggested by genetic studies carried out intransgenic mice (DeChiara et al., 1990, Nature 345:78-80; Louvi et al.,1997, Dev. Biol. 189:33-48). Moreover, when cells transform and becomemalignant, dedifferentiation is often associated with an increased IR-Arelative abundance (Pandini et al., 2002, The Journal of BiologicalChemistry, Vol. 277, No. 42, pp 39684-39695).

Given the high degree of homology, the insulin and IGF-I half-receptors(composed of one α- and one β-subunit) can heterodimerize, leading tothe formation of insulin/IGF-I hybrid receptors (Hybrid-R) (Soos et al.,1990, Biochem J., 270:383-390; Kasuya et al., 1993, Biochemistry32:13531-13536; Seely et al., 1995, Endocrinology, 136:1635-1641;Bailyes et al., 1997, Biochem J., 327:209-215).

Both IR isoforms are equally able to form hybrids with IGF-IR. Hybrid-R,however, have different functional characteristics. Hybrid-RsB hasreduced affinity for IGF1 and especially for IGF2.In contrast,Hybrid-RsA has a high affinity for IGF1 and bind also IGF2 and insulinat a physiological concentration range. The expression of Hybrid-RsAup-regulates the IGF system by two different mechanisms i) binding (withhigh affinity) and activation by both IGF1 and IGF2 (which do not occurwith the Hybrid-RsB), ii) activation of the IGF-IR pathway after insulinbinding. Insulin binding to Hybrid-RsA phosphorylates the IGF-IRβ-subunit and activates an IGF-IR-specific substrate (CrkII) so thatHybrid-RsA shifts insulin to IGF-IR signaling (Pandini et al., 2002).

In several tissues, like liver, spleen or placenta, Hybrid-R are morerepresented than IGF-IR (Bailyes et al., 1997). As tumor tissuesoverexpress, or present an abnormal activation, both IGF-IR and IR-A(Frasca et al., 1999; Sciacca et al., 1999, Oncogene 18:2471-2479; Vellaet al., 2001, Mol. Pathol., 54:121-124), Hybrid-RsA may also beoverexpressed in a variety of human malignancies, including thyroid andbreast cancers providing a selective growth advantage to malignant cellsable to respond by a type IGF-IR signalisation following a stimulationby IGF1 and/or IGF2 but also by insulin at physiological concentrations(Bailyes et al., 1997; Pandini et al., 1999, Clin. Cancer Res.5:1935-1944; Belfiore et al., 1999, Biochimie (Paris) 81:403-407; Frascaet al., 1999; Sciacca et al., 1999; Vella et al., 2001).

The realization of such “therapeutic tools” able to block in the sametime the two receptors is of particular interest as they will allow toavoid the escape phenomena mediated by the expression, or abnormalactivation, in a same tumor of IGF-IR and hybrid-R.

The present invention allows to jointly block the hybrid-R and IGF-IRactivity by generating a compound, and more particularly an antibody, ofhigh affinity able to bind to said two receptors and also to block theiractivation by IGF1, IGF2 or Insulin.

The present invention also deals with the use of an isolated antibodyaccording to the present invention, or a fragment thereof, said antibodyor fragment being able to bind to i) human IGF-IR, and/or to inhibit thebinding of its native ligands, preferably IGF1 and/or IGF2, and/or alsoable to inhibit specifically the tyrosine kinase activity of said IGF-IRand/or ii) hybrid-R, and/or to inhibit the binding of its nativeligands, preferably IGF1, IGF2 and/or Insulin, and/or also able tospecifically inhibit the tyrosine kinase activity of said hybrid-R.

More particularly, in a preferred embodiment, said antibody ischaracterized in that it comprises the sequences of the 7C10 and h7C10antibodies anti-IGF-IR, and fragment thereof, of the present invention,notably the antibodies anti-IGF-IR according to the present inventionhaving a light chain comprising at least a CDR region selected in thegroup consisting in SEQ ID NO: 2, 4 or 6 (or at least a CDR with atleast 80% of homology after optimal alignment with SEQ ID NO: 2, 4 or6), and/or a heavy chain comprising at least a CDR region selected inthe group consisting in SEQ ID NO: 8, 10 or 12 (or at least a CDR withat least 80% of homology after optimal alignment with SEQ ID NO: 8, 10or 12).

According to another preferred embodiment, said antibody is used forcancer therapy, more particularly breast cancer therapy.

Actually, it is known that breast tumoral cells specifically present ontheir surface IGF-IR but also a great number of Insulin receptor and, asa consequence, a great number of Hybrid-R (Frasca et al., 1999; Sciaccaet al., 1999; Vella et al., 2001).

The object of the present invention is to be able to have available amurine monoclonal antibody, preferably a chimerized or humanizedantibody, which will recognize IGF-IR and/or hybrid-R specifically andwith great affinity. This antibody will interact little or not at allwith the IR on insulin. Its attachment will be able to inhibit in vitrothe growth of tumors expressing, or with an abnormal activation of,IGF-IR and/or hybrid-R by interacting principally with the signaltransduction pathways activated during interactions between i) IGF-IRand its native ligands, preferably IGF1 and IGF2 and/or ii) hybrid-R andits native ligands, preferably IGF1, IGF2 and insulin. This antibodywill be able to be active in vivo on all the types of tumors expressing,or with an abnormal activation of, IGF-IR and/or hybrid-R includingestrogen-dependent tumors of the breast and tumors of the prostate,which is not the case for the anti-IGF-IR monoclonal antibodies (writtenMAb or MAB) currently available. In effect, αIR3, which refers to thedomain of IGF-IR, totally inhibits the growth of estrogen-dependenttumors of the breast (MCF-7) in vitro but is without effect on thecorresponding model in vivo (Arteaga C. et al., J. Clin. Invest.,84:1418-1423, 1989). In the same way, the scFv-Fc fragment derived fromthe murine monoclonal 1H7 is only weakly active on the tumor of thebreast MCF-7 and totally inactive on an androgen-independent tumor ofthe prostate (Li S. L. et al., Cancer Immunol. Immunother., 49:243-252,2000). None of these known antibodies are described as being able torecognize, or inhibit the tyrosine kinase activity, of the hybrid-R.

In a surprising manner, the inventors have demonstrated a chimericantibody (called C7C10) and two humanized antibodies respectively calledh7C10 humanized form 1 and h7C10 humanized form 2, derivatives of themurine monoclonal antibody 7C10, recognizing IGF-IR and/or hybrid-R andcorresponding to all of the criteria stated above, that is to say to anonrecognition of the receptor on the insulin, to an in vitro blockageof the IGF1 and/or IGF2 proliferation induced but likewise to the invivo inhibition of the growth of different tumors expressing, or with anabnormal activation of, IGF-IR and/or hybrid-R among which are anosteosarcoma, a non-small cell lung tumor and a pancreatic tumor BxPC3but likewise and more particularly the estrogen-dependent tumor of thebreast MCF-7 and an androgen-independent tumor of the prostate DU-145.Inthe same way, and in a surprising manner, the intensity of inhibition ofthe tumor growth of MCF-7 cells in vivo by the antibody 7C10 iscomparable, or even significantly superior, to that observed withtamoxifen, one of the reference compounds in the treatment ofestrogen-dependent tumors of the breast. Furthermore, it has been shownthat these antibodies inhibit the phosphorylation of the tyrosine of thebeta chain of IGF-IR and of IRS1, the first substrate of the receptor.Moreover, it has likewise been established that these antibodies causethe internalization of said receptor and its degradation contrary towhat is usually observed with natural ligands which allow the rapidrecycling of the receptor on the surface of the cells. It has beenpossible to characterize these antibodies by their peptidic and nucleicsequence, especially by the sequence of their regions determining theircomplementarity (CDR) for IGF-IR and/or hybrid-R.

As the compound is preferably of IgG1 isotype, other mechanisms ofaction involving effector functions, such as for example ADCC and/orCDC, could explain the in vivo efficacy of the antibody of the presentinvention.

Thus, according to a first embodiment, a subject of the presentinvention is an isolated antibody, or one of its functional fragments,said antibody or one of its said fragments being capable of bindingspecifically to the human IGF-IR and/or hybrid-R and, if necessary,preferably moreover capable of inhibiting the binding of the nativeligands preferably IGF1 and/or IGF2 for IGF-IR and IGF1, IGF2 and/orinsulin for hybrid-R and/or capable of specifically inhibiting thetyrosine kinase activity of said IGF-IR and/or hybrid-R, characterizedin that it comprises a light chain comprising at least onecomplementarity determining region CDR chosen from the CDRs of aminoacid sequence SEQ ID Nos. 2, 4 or 6, or at least one CDR whose sequencehas at least 80%, preferably 85%, 90%, 95% and 98% identity, afteroptimum alignment, with the sequence SEQ ID Nos. 2, 4 or 6, or in thatit comprises a heavy chain comprising at least one CDR chosen from theCDRs of amino acid sequence SEQ ID Nos. 8, 10 and 12, or at least oneCDR whose sequence has at least 80%, preferably 85%, 90%, 95% and 98%identity, after optimum alignment, with the sequence SEQ ID NO: 8, 10and 12.

In the present description, the terms “to bind” and “to attach” have thesame meaning and are inter-changeable.

In the present description, the terms polypeptides, polypeptidesequences, peptides and proteins attached to antibody compounds or totheir sequence are interchangeable.

It must be understood here that the invention does not relate to theantibodies in natural form, that is to say they are not in their naturalenvironment but that they have been able to be isolated or obtained bypurification from natural sources, or else obtained by geneticrecombination, or by chemical synthesis, and that they can then containunnatural amino acids as will be described further on.

By CDR region or CDR, it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulins as definedby Kabat et al., (Kabat et al., Sequences of proteins of immunologicalinterest, 5th Ed., U.S. Department of Health and Human Services, NIH,1991, and later editions). 3 heavy chain CDRs and 3 light chain CDRsexist. The term CDR or CDRs is used here in order to indicate, accordingto the case, one of these regions or several, or even the whole, ofthese regions which contain the majority of the amino acid residuesresponsible for the binding by affinity of the antibody for the antigenor the epitope which it recognizes.

By “percentage of identity” between two nucleic acid or amino acidsequences in the sense of the present invention, it is intended toindicate a percentage of nucleotides or of identical amino acid residuesbetween the two sequences to be compared, obtained after the bestalignment (optimum alignment), this percentage being purely statisticaland the differences between the two sequences being distributed randomlyand over their entire length. The comparisons of sequences between twonucleic acid or amino acid sequences are traditionally carried out bycomparing these sequences after having aligned them in an optimummanner, said comparison being able to be carried out by segment or by“comparison window”. The optimum alignment of the sequences for thecomparison can be carried out, in addition to manually, by means of thelocal homology algorithm of Smith and Waterman (1981) [Ad. App. Math.2:482], by means of the local homology algorithm of Neddleman and Wunsch(1970) [J. Mol. Biol. 48: 443], by means of the similarity search methodof Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), bymeans of computer software using these algorithms (GAP, BESTFIT, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis., or else by BLAST N or BLAST Pcomparison software).

The percentage of identity between two nucleic acid or amino acidsequences is determined by comparing these two sequences aligned in anoptimum manner and in which the nucleic acid or amino acid sequence tobe compared can comprise additions or deletions with respect to thereference sequence for an optimum alignment between these two sequences.The percentage of identity is calculated by determining the number ofidentical positions for which the nucleotide or the amino acid residueis identical between the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 in order to obtainthe percentage of identity between these two sequences.

For example, it is possible to use the BLAST program, “BLAST 2sequences” (Tatusova et al., “Blast 2 sequences—a new tool for comparingprotein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250),the parameters used being those given by default (in particular for theparameters “open gap penalty”: 5, and “extension gap penalty”: 2; thematrix chosen being, for example, the matrix “BLOSUM 62” proposed by theprogram), the percentage of identity between the two sequences to becompared being calculated directly by the program.

By amino acid sequence having at least 80%, preferably 85%, 90%, 95% and98% identity with a reference amino acid sequence, those having, withrespect to the reference sequence, certain modifications, in particulara deletion, addition or substitution of at least one amino acid, atruncation or an elongation are preferred. In the case of a substitutionof one or more consecutive or nonconsecutive amino acid(s), thesubstitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. The expression “equivalent aminoacids” is aimed here at indicating any amino acid capable of beingsubstituted with one of the amino acids of the base structure without,however, essentially modifying the biological activities of thecorresponding antibodies and such as will be defined later, especiallyin the examples.

These equivalent amino acids can be determined either by relying ontheir structural homology with the amino acids which they replace, or onresults of comparative trials of biological activity between thedifferent antibodies capable of being carried out.

By way of example, mention is made of the possibilities of substitutioncapable of being carried out without resulting in a profoundmodification of the biological activity of the corresponding modifiedantibody. It is thus possible to replace leucine by valine orisoleucine, aspartic acid by glutamic acid, glutamine by asparagine,arginine by lysine, etc., the reverse substitutions being naturallyenvisageable under the same conditions.

The antibodies according to the present invention are preferablyspecific monoclonal antibodies, especially of murine, chimeric orhumanized origin, which can be obtained according to the standardmethods well known to the person skilled in the art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 726, 1988) or tothe technique of preparation from hybridomas described by Kohler andMilstein (Nature, 256:495-497, 1975).

The monoclonal antibodies according to the invention can be obtained,for example, from an animal cell immunized against the IGF-IR and/orhybrid-R, or one of their fragments containing the epitope specificallyrecognized by said monoclonal antibodies according to the invention.Said IGF-IR and/or hybrid-R, or one of their said fragments, canespecially be produced according to the usual working methods, bygenetic recombination starting with a nucleic acid sequence contained inthe cDNA sequence coding for the IGF-IR and/or hybrid-R or by peptidesynthesis starting from a sequence of amino acids comprised in thepeptide sequence of the IGF-IR and/or hybrid-R.

The monoclonal antibodies according to the invention can, for example,be purified on an affinity column on which the IGF-IR and/or hybrid-R orone of their fragments containing the epitope specifically recognized bysaid monoclonal antibodies according to the invention has previouslybeen immobilized. More particularly, said monoclonal antibodies can bepurified by chromatography on protein A and/or G, followed or notfollowed by ion-exchange chromatography aimed at eliminating theresidual protein contaminants as well as the DNA and the LPS, in itselffollowed or not followed by exclusion chromatography on Sepharose gel inorder to eliminate the potential aggregates due to the presence ofdimers or of other multimers. In an even more preferred manner, thewhole of these techniques can be used simultaneously or successively.Such a purification can be done in the same time or sequenced with firsta recognition on one receptor selected between IGF-IR or hybrid-R and,in a second step, the recognition of the other one.

Chimeric or humanized antibodies are likewise included in antibodiesaccording to the present invention.

By chimeric antibody, it is intended to indicate an antibody whichcontains a natural variable (light chain and heavy chain) region derivedfrom an antibody of a given species in combination with the light chainand heavy chain constant regions of an antibody of a speciesheterologous to said given species.

The antibodies or their fragments of chimeric type according to theinvention can be prepared by using the techniques of geneticrecombination. For example, the chimeric antibody can be produced bycloning a recombinant DNA containing a promoter and a sequence codingfor the variable region of a nonhuman, especially murine, monoclonalantibody according to the invention and a sequence coding for theconstant region of human antibody. A chimeric antibody of the inventionencoded by such a recombinant gene will be, for example, a mouse-manchimera, the specificity of this antibody being determined by thevariable region derived from the murine DNA and its isotype determinedby the constant region derived from the human DNA. For the methods ofpreparation of chimeric antibodies, it is possible, for example, torefer to the document Verhoeyn et al., (BioEssays, 8:74, 1988).

By humanized antibody, it is intended to indicate an antibody whichcontains CDR regions derived from an antibody of nonhuman origin, theother parts of the antibody molecule being derived from one (or fromseveral) human antibodies. Moreover, some of the residues of thesegments of the skeleton (called FR) can be modified in order toconserve the affinity of the binding (Jones et al., Nature, 321:522-525,1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al.,Nature, 332:323-327, 1988).

The humanized antibodies according to the invention or their fragmentscan be prepared by techniques known to the person skilled in the art(such as, for example, those described in the documents Singer et al.,J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng.Rev., 10: 1-142, 1992; or Bebbington et al., Bio/Technology, 10:169-175,1992). Such humanized antibodies according to the invention arepreferred for their use in in vitro diagnostic methods, or in vivoprophylactic and/or therapeutic treatment.

By functional fragment of an antibody according to the invention, it isintended to indicate in particular an antibody fragment, such as Fv,scFv (sc for single chain), Fab, F(ab′)₂, Fab′, scFv-Fc fragments ordiabodies, or any fragment of which the half-life time would have beenincreased by chemical modification, such as the addition ofpoly(alkylene)glycol such as poly(ethylene)glycol (“PEGylation”)(pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂-PEG orFab′-PEG) (“PEG” for Poly(Ethylene) Glycol), or by incorporation in aliposome, said fragments having at least one of the characteristic CDRsof sequence SEQ ID NO: 2, 4, 6, 8, 10 or 12 according to the invention,and, especially, in that it is capable of exerting in a general manneran even partial activity of the antibody from which it is descended,such as in particular the capacity to recognize and to bind to theIGF-IR receptor, and, if necessary, to inhibit the activity of theIGF-IR and/or hybrid-R.

Preferably, said functional fragments will be constituted or willcomprise a partial sequence of the heavy or light variable chain of theantibody from which they are derived, said partial sequence beingsufficient to retain the same specificity of binding as the antibodyfrom which it is descended and a sufficient affinity, preferably atleast equal to 1/100, in a more preferred manner to at least 1/10, ofthat of the antibody from which it is descended, with respect to theIGF-IR and/or hybrid-R.

Such a functional fragment will contain at the minimum 5 amino acids,preferably 10, 15, 25, 50 and 100 consecutive amino acids of thesequence of the antibody from which it is descended.

Preferably, these functional fragments will be fragments of Fv, scFv,Fab, F(ab′)₂, F(ab′), scFv-Fc type or diabodies, which generally havethe same specificity of binding as the antibody from which they aredescended. According to the present invention, antibody fragments of theinvention can be obtained starting from antibodies such as describedabove by methods such as digestion by enzymes, such as pepsin or papainand/or by cleavage of the disulfide bridges by chemical reduction. Inanother manner, the antibody fragments comprised in the presentinvention can be obtained by techniques of genetic recombinationlikewise well known to the person skilled in the art or else by peptidesynthesis by means of, for example, automatic peptide synthesizers suchas those supplied by the company Applied Biosystems, etc.

In a more preferred manner, the invention comprises the antibodies, ortheir functional fragments, according to the present invention,especially chimeric or humanized antibodies, obtained by geneticrecombination or by chemical synthesis.

In a preferred embodiment, a subject of the invention is an antibody, orone of its functional fragments, according to the invention,characterized in that it comprises a heavy chain comprising at least oneCDR chosen from the CDRs of sequence SEQ ID NO: 8, 10 or 12 or asequence having at least 80% identity after optimum alignment with thesequence SEQ ID NO: SEQ ID NO: 8, 10 or 12.

Among the six short CDR sequences, the third CDR of the heavy chain(CDRH3) has a greater size variability (greater diversity essentiallydue to the mechanisms of arrangement of the genes which give rise toit). It can be as short as 2 amino acids although the longest size knownis 26. Functionally, CDRH3 plays a role in part in the determination ofthe specificity of the antibody (Segal et al., PNAS, 71:4298-4302, 1974;Amit et al., Science, 233:747-753, 1986; Chothia et al., J. Mol. Biol.,196:901-917, 1987; Chothia et al., Nature, 342:877-883, 1989; Caton etal., J. Immunol., 144:1965-1968, 1990; Sharon et al., PNAS,87:4814-4817, 1990; Sharon et al., J. Immunol., 144:4863-4869, 1990;Kabat et al., J. Immunol., 147:1709-1719, 1991).

It is known that only a low percentage of the amino acids of the CDRscontribute to the construction of an antibody binding site, but theseresidues must be maintained in a very specific tridimensionalconformation.

In a more preferred manner, the present invention relates to an antibodyor one of its functional fragments, according to the invention,characterized in that it comprises a heavy chain comprising at least twoof the three CDRs or the three CDRs of sequence SEQ ID Nos. 8, 10 and12, or at least two of three CDRs or three CDRs of sequence respectivelyhaving at least 80% identity after optimum alignment with the sequenceSEQ ID NO: 8, 10 and 12.

In a likewise preferred embodiment, a subject of the invention is anantibody or one of its functional fragments, according to the invention,characterized in that it comprises a light chain comprising at least oneCDR chosen from the CDRs of sequence SEQ ID NO: 2, 4 or 6, or a CDRwhose sequence has at least 80% identity after optimum alignment withthe sequence SEQ ID NO: 2, 4 or 6.

In a more preferred embodiment, a subject of the invention is anantibody or one of its functional fragments according to the invention,characterized in that it comprises a light chain comprising at least twoof the three CDRs or the three CDRs of sequence SEQ ID Nos. 2, 4 and 6,or at least two of three CDRs or three CDRs of sequence respectivelyhaving at least 80% identity after optimum alignment with the sequenceSEQ ID NO: 2, 4 and 6.

In a more preferred manner, the antibody or one of its functionalfragments according to the invention is characterized in that itcomprises a heavy chain comprising the three CDRs of sequence SEQ IDNos. 8, 10 and 12, or three CDRs of sequence respectively having atleast 80% of identity after optimum alignment with the sequence SEQ IDNO: 8, 10 and 12 and in that it moreover comprises a light chaincomprising the three CDRs of sequence SEQ ID Nos. 2, 4 and 6, or threeCDRs of sequence respectively having at least 80% of identity afteroptimum alignment with the sequence SEQ ID NO: 2, 4 and 6.

According to another aspect, a subject of the present invention is anantibody or one of its functional fragments, according to the invention,characterized in that it does not attach or it does not attach in asignificant manner to the human insulin receptor IR.

In a preferred manner, said functional fragments according to thepresent invention will be chosen from the fragments Fv, scFv, Fab,(Fab′)₂, Fab′, scFv-Fc or diabodies, or any functional fragment whosehalf-life would have been increased by a chemical modification,especially by PEGylation, or by incorporation in a liposome.

According to another aspect, the invention relates to a murine hybridomacapable of secreting a monoclonal antibody according to the presentinvention, especially the hybridoma of murine origin such as depositedat the Centre National de Culture De Microorganisme (CNCM, NationalCenter of Microorganism Culture) (Institut Pasteur, Paris, France) onSep. 19, 2001 under the number 1-2717.

The monoclonal antibody here called 7C10, or one of its functionalfragments, characterized in that said antibody is secreted by thehybridoma deposited at the CNCM on Sep. 19, 2001 under the number 1-2717is, of course, part of the present invention.

In a particular embodiment, the present invention relates to a murineantibody, or one of its functional fragments, according to theinvention, characterized in that said antibody comprises a light chainof sequence comprising the amino acid sequence SEQ ID NO: 54, or asequence having at least 80% identity after optimum alignment with thesequence SEQ ID NO: 54, or/and in that it comprises a heavy chain ofsequence comprising the amino acid sequence SEQ ID NO: 69, or a sequencehaving at least 80% identity after optimum alignment with the sequenceSEQ ID NO: 69.

According to a likewise particular aspect, the present invention relatesto a chimeric antibody, or one of its functional fragments, according tothe invention, characterized in that said antibody moreover comprisesthe light chain and heavy chain constant regions derived from anantibody of a species heterologous to the mouse, especially man, and ina preferred manner in that the light chain and heavy chain constantregions derived from a human antibody are respectively the kappa andgamma-1, gamma-2 or gamma-4 region.

According to a likewise particular aspect, the present invention relatesto a humanized antibody or one of its functional fragments, according tothe invention, characterized in that said antibody comprises a lightchain and/or a heavy chain in which the skeleton segments FR1 to FR4(such as defined below in examples 12 and 13, in tables 5 and 6) of saidlight chain and/or heavy chain are respectively derived from skeletonsegments FR1 to FR4 of human antibody light chain and/or heavy chain.

According to a preferred embodiment, the humanized antibody or one ofits functional fragments, according to the present invention ischaracterized in that said humanized antibody comprises a light chaincomprising the amino acid sequence SEQ ID NO: 61 or 65, or a sequencehaving at least 80% identity after optimum alignment with the sequenceSEQ ID NO: 61 or 65, or/and in that it comprises a heavy chaincomprising the amino acid sequence SEQ ID NO: 75, 79 or 83, or asequence having at least 80% identity after optimum alignment with thesequence SEQ ID NO: 75, 79 or 83.

Preferably, the humanized antibody, or one of its functional fragments,according to the invention is characterized in that said humanizedantibody comprises a light chain comprising the amino acid sequence SEQID NO: 65, and in that it comprises a heavy chain of sequence comprisingthe amino acid sequence SEQ ID NO: 79 or 83, preferably SEQ ID NO: 83.

According to a novel aspect, the present invention relates to anisolated nucleic acid, characterized in that it is chosen from thefollowing nucleic acids:

a) a nucleic acid, DNA or RNA, coding for an antibody, or one of itsfunctional fragments, according to the invention;

b) a complementary nucleic acid of a nucleic acid such as defined in a);and

c) a nucleic acid of at least 18 nucleotides capable of hybridizingunder conditions of great stringency with at least one of the CDRs ofnucleic acid sequence SEQ ID NO: 1, 3, 5, 7, 9 or 11, or with a sequencehaving at least 80%, preferably 85%, 90%, 95% and 98%, identity afteroptimum alignment with the sequence SEQ ID NO: 1, 3, 5, 7, 9 or 11.

By nucleic acid, nucleic or nucleic acid sequence, polynucleotide,oligonucleotide, polynucleotide sequence, nucleotide sequence, termswhich will be employed indifferently in the present invention, it isintended to indicate a precise linkage of nucleotides, which aremodified or unmodified, allowing a fragment or a region of a nucleicacid to be defined, containing or not containing unnatural nucleotides,and being able to correspond just as well to a double-stranded DNA, asingle-stranded DNA as to the transcription products of said DNAs.

It must also be understood here that the present invention does notconcern the nucleotide sequences in their natural chromosomalenvironment, that is to say in the natural state. It concerns sequenceswhich have been isolated and/or purified, that is to say that they havebeen selected directly or indirectly, for example by copy, theirenvironment having been at least partially modified. It is thus likewiseintended to indicate here the isolated nucleic acids obtained by geneticrecombination by means, for example, of host cells or obtained bychemical synthesis.

By nucleic sequences having a percentage of identity of at least 80%,preferably 85%, 90%, 95% and 98%, after optimum alignment with apreferred sequence, it is intended to indicate the nucleic sequenceshaving, with respect to the reference nucleic sequence, certainmodifications such as, in particular, a deletion, a truncation, anelongation, a chimeric fusion and/or a substitution, especially pointsubstitution. It preferably concerns sequences in which the sequencescode for the same amino acid sequences as the reference sequence, thisbeing connected to the degeneracy of the genetic code, or complementarysequences which are capable of hybridizing specifically with thereference sequences, preferably under conditions of high stringency,especially such as defined below.

A hybridization under conditions of high stringency signifies that thetemperature conditions and ionic strength conditions are chosen in sucha way that they allow the maintenance of the hybridization between twofragments of complementary DNA. By way of illustration, conditions ofhigh stringency of the hybridization step for the purposes of definingthe polynucleotide fragments described above are advantageously thefollowing.

The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH7.5) containing 5×SSC (1×SSC corresponds to a 0.15 M NaCl+0.015 M sodiumcitrate solution), 50% of formamide, 7% of sodium dodecyl sulfate (SDS),10× Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2)actual hybridization for 20 hours at a temperature dependent on the sizeof the probe (i.e., 42° C., for a probe size >100 nucleotides) followedby 2 washes of 20 minutes at 20° C. in 2×SSC+2% of SDS, 1 wash of 20minutes at 20° C. in 0.1×SSC+0.1% of SDS. The last wash is carried outin 0.1×SSC+0.1% of SDS for 30 minutes at 60° C. for a probe size >100nucleotides. The hybridization conditions of high stringency describedabove for a polynucleotide of defined size can be adapted by the personskilled in the art for oligonucleotides of greater or smaller size,according to the teaching of Sambrook et al., (1989, Molecular cloning:a laboratory manual. 2nd Ed. Cold Spring Harbor).

The invention likewise relates to a vector comprising a nucleic acidaccording to the present invention.

The invention aims especially at cloning and/or expression vectors whichcontain a nucleotide sequence according to the invention.

The vectors according to the invention preferably contain elements whichallow the expression and/or the secretion of the nucleotide sequences ina determined host cell. The vector must therefore contain a promoter,signals of initiation and termination of translation, as well asappropriate regions of regulation of transcription. It must be able tobe maintained in a stable manner in the host cell and can optionallyhave particular signals which specify the secretion of the translatedprotein. These different elements are chosen and optimized by the personskilled in the art as a function of the host cell used. To this effect,the nucleotide sequences according to the invention can be inserted intoautonomous replication vectors in the chosen host, or be integrativevectors of the chosen host.

Such vectors are prepared by methods currently used by the personskilled in the art, and the resulting clones can be introduced into anappropriate host by standard methods, such as lipofection,electroporation, thermal shock, or chemical methods.

The vectors according to the invention are, for example, vectors ofplasmidic or viral origin. They are useful for transforming host cellsin order to clone or to express the nucleotide sequences according tothe invention.

The invention likewise comprises the host cells transformed by orcomprising a vector according to the invention.

The host cell can be chosen from prokaryotic or eukaryotic systems, forexample bacterial cells but likewise yeast cells or animal cells, inparticular mammalian cells. It is likewise possible to use insect cellsor plant cells.

The invention likewise concerns animals, except man, which comprise atleast one cell transformed according to the invention.

According to another aspect, a subject of the invention is a process forproduction of an antibody, or one of its functional fragments accordingto the invention, characterized in that it comprises the followingstages:

a) culture in a medium and appropriate culture conditions of a host cellaccording to the invention; and

b) the recovery of said antibodies, or one of their functionalfragments, thus produced starting from the culture medium or saidcultured cells.

The cells transformed according to the invention can be used inprocesses for preparation of recombinant polypeptides according to theinvention. The processes for preparation of a polypeptide according tothe invention in recombinant form, characterized in that they employ avector and/or a cell transformed by a vector according to the invention,are themselves comprised in the present invention. Preferably, a celltransformed by a vector according to the invention is cultured underconditions which allow the expression of said polypeptide and saidrecombinant peptide is recovered.

As has been said, the host cell can be chosen from prokaryotic oreukaryotic systems. In particular, it is possible to identify nucleotidesequences according to the invention, facilitating secretion in such aprokaryotic or eukaryotic system. A vector according to the inventioncarrying such a sequence can therefore advantageously be used for theproduction of recombinant proteins, intended to be secreted. In effect,the purification of these recombinant proteins of interest will befacilitated by the fact that they are present in the supernatant of thecell culture rather than in the interior of the host cells.

It is likewise possible to prepare the polypeptides according to theinvention by chemical synthesis. Such a preparation process is likewisea subject of the invention. The person skilled in the art knows theprocesses of chemical synthesis, for example the techniques employingsolid phases (see especially Steward et al., 1984, Solid phase peptidesynthesis, Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)) ortechniques using partial solid phases, by condensation of fragments orby a classical synthesis in solution. The polypeptides obtained bychemical synthesis and being able to contain corresponding unnaturalamino acids are likewise comprised in the invention.

The antibodies, or one of their functional fragments, capable of beingobtained by a process according to the invention are likewise comprisedin the present invention.

According to a second embodiment, the present invention concerns anantibody according to the invention such as described further above,characterized in that it is, moreover, capable of binding specificallyto the human epidermal growth factor receptor EGFR and/or capable ofspecifically inhibiting the tyrosine kinase activity of said EGFRreceptor.

In a general manner, the growth factors are small proteins involved inthe regulation of the proliferation and of the differentiation of normalcells. Some of these growth factors likewise play an important role inthe initiation and the maintenance of cell transformation, being able tofunction as autocrine or paracrine factors. This is especially the case,in addition to the IGF1 described further above, for the epidermalgrowth factor EGF, which seems particularly involved in the appearanceof the tumor phenotype, the progression of tumors and the generation ofmetastases.

EGF and IGF1 exert their action through the intermediary of theirrespective receptor here called EGFR and IGF-IR and/or hybrid-R. Itconcerns in the two cases membrane receptors with tyrosine kinaseactivity whose overexpression is described in numerous cancers. It must,however, be noted that the interaction of these two receptors is notclearly established and that the studies carried out by various teams inthis connection give contradictory results as to the collaboration ofthese two receptors.

Studies carried out on prostate tumor cells show that the interruptionof the autocrine loop EGF/EGFR by an anti-EGFR monoclonal antibody (herecalled “MAB” or “MAb”) is manifested by a complete loss of the responseof the DU145 cells to IGF1 (Connolly J. M. and Rose D. P., Prostate,Apr. 24 (4):167-75, 1994; Putz T. et al., Cancer Res., Jan. 1,59(1):227-33, 1999). These results would suggest that a blockage of thereceptor for the EGF would be sufficient in order to obtain a totalinhibition of the transformation signals generated by the activation ofthe three receptors (EGFR and IGF-IR and hybrid-R). On the other hand,other studies (Pietrzkowski et al., Cell Growth Differ, Apr. 3(4):199-205, 1992; Coppola et al., Mol Cell Biol., Jul. 14 (7):4588-95,1994) have shown that an over-expression of EGFR necessitates thepresence of a functional IGF-IR in order to exert its mitogenic andtransformant potential, although IGF-IR does not necessitate, for itspart, the presence of functional EGFR in order to mediate its action.This second series of studies would be more in agreement with a strategytending preferentially to block IGF-IR with the aim of simultaneouslyaffecting the two receptors.

In a surprising manner, the inventors have, firstly, demonstrated that aco-inhibition of the attachment of the IGF1 and/or IGF2 to the IGF-IRand/or of the IGF1 and/or IGF2 and/or insulin to the hybrid-R and of theattachment of the EGF to the EGFR allows a significant synergy of actionof these two actions to be obtained against the in vivo tumor growth innude mice carrying a tumor expressing these two receptors. One of themore probable hypotheses which is able to explain this synergy of actionis that the two growth factors EGF and IGF1 (and/or IGF2) themselves actin synergy in the transformation of normal cells to cells with tumoralcharacter and/or in the growth and/or the proliferation of tumor cellsfor certain tumors, especially for those overexpressing, or with anabnormal activation of, the three receptors EGFR, IGF-IR and hybrid-Rand/or having an overactivation of the transduction signal mediated bythese three receptors, in particular at the level of the tyrosine kinaseactivity of these receptors.

According to a preferred aspect of this embodiment, the inventionconcerns an antibody such as described further above, characterized inthat it consists of a bispecific antibody comprising a second motifspecifically inhibiting the attachment of the EGF to the EGFR and/orspecifically inhibiting the tyrosine kinase activity of said EGFR.

The term “second motif” is intended to indicate above especially asequence of amino acids comprising a fragment capable of specificallybinding to EGFR, in particular a CDR region of a variable chain of ananti-EGFR antibody, or one of the fragments of this CDR region ofsufficient length in order to exert this specific binding, or elseseveral CDR regions of an anti-EGFR antibody.

The bispecific or bifunctional antibodies form a second generation ofmonoclonal antibodies in which two different variable regions arecombined in the same molecule (Hollinger and Bohlen 1999 Cancer andmetastasis rev. 18: 411-419). Their use has been demonstrated both inthe diagnostic field and in the therapy field from their capacity torecruit new effector functions or to target several molecules on thesurface of tumor cells. These antibodies can be obtained by chemicalmethods (Glennie M J et al., 1987 J. Immunol. 139, 2367-2375; Repp R. etal., 1995 J. Hemat. 377-382) or somatic methods (Staerz U. D. and BevanM. J. 1986 PNAS 83, 1453-1457; Suresh M. R. et al., 1986 Method Enzymol.121: 210-228) but likewise and preferentially by genetic engineeringtechniques which allow the heterodimerization to be forced and thusfacilitate the process of purification of the antibody sought (Merchandet al., 1998 Nature Biotech. 16:677-681).

These bispecific antibodies can be constructed as entire IgG, asbispecific Fab′2, as Fab′PEG or as diabodies or else as bispecific scFvbut likewise as a tetravalent bispecific antibody or two attachmentsites are present for each antigen targeted (Park et al., 2000 Mol.Immunol. 37 (18):1123-30) or its fragments as described further above.

In addition to an economic advantage from the fact that the productionand the administration of a bispecific antibody are less onerous thanthe production of two specific antibodies, the use of such bispecificantibodies has the advantage of reducing the toxicity of the treatment.This is because the use of a bispecific antibody allows the totalquantity of circulating antibodies to be reduced and, consequently, thepossible toxicity.

In a preferred embodiment of the invention, the bispecific antibody is abivalent or tetravalent antibody.

In practice, the interest in using a tetravalent bispecific antibody isthat it has a greater avidity in comparison with a bivalent antibody onaccount of the presence of three attachment sites for each target,respectively IGF-IR, hybrid-R and EGFR in the present invention.

In a similar manner to the selection of the functional fragments of theanti-IGF-IR and/or hybrid-R antibody described above, said second motifis selected from the fragments Fv, Fab, F(ab′)₂, Fab′, scFv, scFv-Fc andthe diabodies, or any form whose half-life would have been increasedlike the pegylated fragments such as Fv-PEG, scFv-PEG, Fab-PEG,F(ab′)₂-PEG or Fab′-PEG. According to an even more preferred aspect ofthe invention, said second anti-EGFR motif is descended from the mousemonoclonal antibody 225, its mouse-man chimeric derivative C225, or ahumanized antibody derived from this antibody 225.

According to yet another aspect, a subject of the invention is anantibody, or one of its functional fragments, according to the inventionas a medicament, preferably a humanized antibody such as defined above.Antibody, for the remainder of the present description, must beunderstood as an anti-IGF-IR and/or hybrid-R antibody as well as abispecific anti-IGF-IR and/or hybrid-R/EGFR antibody.

The invention likewise concerns a pharmaceutical composition comprisingby way of active principle a compound consisting of an antibody, or oneof its functional fragments according to the invention, preferably mixedwith an excipient and/or a pharmaceutically acceptable vehicle.

According to yet another embodiment, the present invention likewiseconcerns a pharmaceutical composition such as described further abovewhich comprises a second compound chosen from the compounds capable ofspecifically inhibiting the attachment of the EGF to the human epidermalgrowth factor receptor EGFR and/or capable of specifically inhibitingthe tyrosine kinase activity of said EGFR.

In a preferred aspect of the invention, said second compound is chosenfrom the isolated anti-EGFR antibodies, or their functional fragments,capable of inhibiting by competition the attachment of the EGF to theEGFR. More particularly, said anti-EGFR antibody is chosen from themonoclonal, chimeric or humanized anti-EGFR antibodies, or theirfunctional fragments.

Even more particularly, said functional fragments of the anti-EGFRantibody are chosen from the fragments Fv, Fab, F(ab′)₂, Fab′, scFv-Fcor diabodies, or any fragment whose half-life would have been increased,like pegylated fragments. Said antibody can consist, in an even morepreferred manner, of the mouse monoclonal antibody 225, its mouse-manchimeric derivative C225 (also called IMC-C225) or a humanized antibodyderived from this antibody 225.

Another complementary embodiment of the invention consists in acomposition such as described above which comprises, moreover, as acombination product for simultaneous, separate or sequential use, acytotoxic/cytostatic agent and/or an inhibitor of the tyrosine kinaseactivity respectively of the receptors for IGF-I and/or for EGF.

“Simultaneous use” is understood as meaning the administration of thetwo compounds of the composition according to the invention in a singleand identical pharmaceutical form.

“Separate use” is understood as meaning the administration, at the sametime, of the two compounds of the composition according to the inventionin distinct pharmaceutical forms.

“Sequential use” is understood as meaning the successive administrationof the two compounds of the composition according to the invention, eachin a distinct pharmaceutical form.

In a general fashion, the composition according to the inventionconsiderably increases the efficacy of the treatment of cancer. In otherwords, the therapeutic effect of the anti-IGF-IR and/or hybrid-Rantibody according to the invention is potentiated in an unexpectedmanner by the administration of a cytotoxic agent. Another majorsubsequent advantage produced by a composition according to theinvention concerns the possibility of using lower efficacious doses ofactive principle, which allows the risks of appearance of secondaryeffects to be avoided or to be reduced, in particular the effects of thecytotoxic agent.

In addition, this composition according to the invention would allow theexpected therapeutic effect to be attained more rapidly.

In a particularly preferred embodiment, said composition as acombination product according to the invention is characterized in thatsaid cytotoxic/cytostatic agent is chosen from the agents interactingwith DNA, the antimetabolites, the topoisomerase I or II inhibitors, orelse the spindle inhibitor or stabilizer agents or else any agentcapable of being used in chemotherapy. Such cytotoxic/cytostatic agents,for each of the aforesaid classes of cytotoxic agents, are, for example,cited in the 2001 edition of VIDAL, on the page devoted to the compoundsattached to the cancerology and hematology column “Cytotoxics”, thesecytotoxic compounds cited with reference to this document are cited hereas preferred cytotoxic agents.

In a particularly preferred embodiment, said composition as acombination product according to the invention is characterized in thatsaid cytotoxic agent is coupled chemically to said antibody forsimultaneous use.

In a particularly preferred embodiment, said composition according tothe invention is characterized in that said cytotoxic/cytostatic agentis chosen from the spindle inhibitor or stabilizer agents, preferablyvinorelbine and/or vinflunine and/or vincristine.

Immunoliposomes are liposomes capable of vehicling compounds, such ascytotoxic and/or cytostatic agents, such as described above, and ofaddressing them to tumor cells by means of antibodies or of antibodyfragments attached to their surface. The antibodies or antibodyfragments used are directed against antigens overexpressed at thesurface of tumor cells and/or surface antigens the expression of whichis restricted to tumor cells. They are preferably directed againsttyrosine kinase receptors, and more particularly against the receptorsfor IGF-I, EGF or else VEGF. A preferred antibody is a monoclonal orpolyclonal, preferably monoclonal, or even humanized, antibody whichwill recognize the IGF-IR and/or hybrid-R specifically and with highaffinity. Even more preferably, this antibody consists of the antibodywhich is the subject of the present invention.

The use of immunoliposomes for inhibiting tumor cell growth has beendescribed in the literature. By way of example, mention may be made ofthe immunoliposomes which target proteins, such as ErbB2 (Hurwitz E. etal., Cancer Immunol. Immunother, 49:226-234, 2000; Park J. W. et al.,Clinical Cancer Res., 8:1172-1181, 2002) or EGFR (Harding J. A. et al.,Biochim. Biophys. Acta, 1327:181-192, 1997), or glycolipids such as theganglioside GD2 (Pastorino F. et al., Cancer Res., 63:86-92, 2003).

Immunoliposomes combine the advantages of liposomes and ofimmunoconjugates. Liposomes in fact make it possible to encapsulatecytotoxic and/or cytostatic agents and thus to protect them againstdegradation. They also have the advantage of decreasing the toxicity ofthe vehiculed agents and of reducing the side effects that they induce.They may thus allow the use of agents which are much more toxic than theagents conventionally used in anticancer chemotherapies. The conjugationof antibodies or of antibody fragments to the surface of liposomes hasthe advantage of thus providing a system for specific targeting andaddressing of the cytotoxic agent encapsulated in the liposome. Inaddition, unlike immunoconjugates, since the vehiculed agent is notcovalently coupled to the antibody or to the antibody fragment, it willbe completely active as soon as it is introduced into the target cell.

The antibodies or antibody fragments may be attached, without anylimitation, covalently to the surface of the liposomes usingconventional methods of bioconjugation. The coupling of these antibodiesor of the fragments will be carried out on the lipids or lipids carryinga PEG which have been inserted into the liposomal membrane. In the caseof a PEG-lipid, the coupling will be carried out on the PEG in thedistal position with respect to the lipid. Liposomes carrying PEG groups(PEG-grafted liposomes) have the advantage of having longer half-livesthan “naked” liposomes. By way of example, mention may be made ofcoupling of the antibody or of the fragment, via thiol groups, to theactivated lipids or PEG-lipids exhibiting maleimide or bromoacetylgroups. The thiol groups for this type of coupling may come from 2sources. They may be free cysteine residues introduced into arecombinant fragment of the antibody of interest, for example Fab′ orscFv fragments with an additional cysteine residue, or released afterenzymatic hydrolysis of the antibody of interest and controlledreduction, which is the case, for example, during the preparation ofFab′ fragments from complete antibodies. Complete antibodies can also becoupled, after controlled oxidation of the oligosaccharides carried bythe heavy chains, to lipids or PEG-lipids exhibiting free amine orhydrazide groups.

Since tumor cells overexpressing, or with an abnormal activation of, theIGF-IR and/or hybrid-R generally possess the property of alsooverexpressing EGFR, it could also prove to be advantageous to claimbispecific immunoliposomes for targeting both the IGF-IR and/or thehybrid-R and the EGFR. Similarly, monospecific liposomes to the surfaceof which would be grafted one of the native ligands for these threereceptors, IGF1, IGF2, insulin or EGF, or bispecific liposomes, wouldmake it possible to target the same tumor cells overexpressing, or withan abnormal activation of, at least one of these receptors. Thisapproach has been described for the EGFR (Kullberg E. B. et al., Pharm.Res., 20:229-236, 2003) but not for the IGF-IR or for the hybrid-R.

Such immunoliposomes having antibodies anti-IGR-IR and/or hybrid-R, orfragments thereof, attached covalently to the surface of the liposomes,are comprised in the present invention.

Method for the treatment of cancer wherein such immunoliposomes areadministrated to patient in need of such treatment, forms also part ofthe present invention.

In order to facilitate the coupling between said cytotoxic agent andsaid antibody according to the invention, it is especially possible tointroduce spacer molecules between the two compounds to be coupled, suchas poly(alkylene) glycols like polyethylene glycol, or else amino acids,or, in another embodiment, to use active derivatives of said cytotoxicagents into which would have been introduced functions capable ofreacting with said antibody according to the invention. These couplingtechniques are well known to the person skilled in the art and will notbe expanded upon in the present description.

In another preferred embodiment, said inhibitor of the tyrosine kinaseactivity of the receptors for IGF-I and/or for EGF is selected from thegroup consisting of derived natural agents, dianilinophthalimides,pyrazolo- or pyrrolopyridopyrimidines or else quinazilines. Suchinhibitory agents are well known to the person skilled in the art anddescribed in the literature (Ciardiello F., Drugs 2000, Suppl. 1,25-32).

Other inhibitors of EGFR can, without any limitation, consist of theanti-EGFR monoclonal antibodies C225 and 22Mab (ImClone SystemsIncorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA) orthe compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI-166(Novartis), PKI-166/CGP-75166 (Novartis), PTK 787 (Novartis), CP 701(Cephalon), leflunomide (Pharmacia/Sugen), CI-1033 (Warner-LambertParke-Davis), CU-1033/PD 183, 805 (Warner-Lambert Parke-Davis), CL-387,785 (Wyeth-Ayerst), BBR-1611 (Boehringer Mannheim GmbH/Roche), NaamidineA (Bristol-Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (BoehringerIngelheim), OLX-103 (Merck & Co), VRCTC-310 (Ventech Research), EGFfusion toxin (Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808(Imperial Cancer Research Fund), RG-50864 (INSERM), LFM-A12 (ParkerHughes Cancer Center), WHI-P97 (Parker Hughes Cancer Center), GW-282974(Glaxo), KT-8391 (Kyowa Hakko) or the “EGFR Vaccine” (YorkMedical/Centro de Immunologia Molecular).

According to yet another embodiment of the invention, the compositionsuch as described above can likewise comprise another antibody compounddirected against the extracellular domain of the HER2/neu receptor, as acombination product for simultaneous, separate or sequential use,intended for the prevention and for the treatment of cancer, especiallythe cancers overexpressing said HER2/neu receptor and the receptorIGF-IR and/or EGFR, such as especially cancer of the breast.

Reference can be made especially to the publications of Albanell et al.,(J. of the National Cancer Institute, 93(24):1830-1831, 2001) and of Luet al., (J. of the National Cancer Institute, 93(24):1852-1857, 2001)justifying the unexpected interest in combining an anti-HER2/neuantibody with an anti-IGF-IR and/or hybrid-R antibody according to thepresent invention.

In a particular manner, said anti-HER2/neu antibody of the compositionaccording to the invention is the antibody called Trastuzumab (alsocalled Herceptin).

The invention relates, in another aspect, to a composition characterizedin that one, at least, of said antibodies, or one of their functionalfragments, is conjugated with a cell toxin and/or a radioelement.

Preferably, said toxin or said radioelement is capable of inhibiting atleast one cell activity of cells expressing, or with an abnormalactivation of, the IGF-IR and/or hybrid-R and/or EGFR, in a morepreferred manner capable of preventing the growth or the proliferationof said cell, especially of totally inactivating said cell.

Preferably also, said toxin is an enterobacterial toxin, especiallyPseudomonas exotoxin A.

The radioelements (or radioisotopes) preferably conjugated to theantibodies employed for the therapy are radioisotopes which emit gammarays and preferably iodine¹³¹, yttrium⁹⁰, gold¹⁹⁹, palladium¹⁰⁰,copper⁶⁷, bismuth²¹⁷ and antimony²¹¹. The radioisotopes which emit betaand alpha rays can likewise be used for the therapy.

By toxin or radioelement conjugated to at least one antibody, or one ofits functional fragments, according to the invention, it is intended toindicate any means allowing said toxin or said radioelement to bind tosaid at least one antibody, especially by covalent coupling between thetwo compounds, with or without introduction of a linking molecule.

Among the agents allowing binding in a chemical (covalent),electrostatic or noncovalent manner of all or part of the components ofthe conjugate, mention may particularly be made of benzoquinone,carbodiimide and more particularly EDC(1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride),dimaleimide, dithiobis-nitrobenzoic acid (DTNB), N-succinimidyl S-acetylthio-acetate (SATA), the bridging agents having one or more phenylazidegroups reacting with the ultraviolets (U.V.) and preferablyN-[-4-(azidosalicylamino)butyl]-3′-(2′-pyridyldithio)propionamide(APDP), N-succinimid-yl 3-(2-pyridyldithio)propionate (SPDP),6-hydrazino-nicotinamide (HYNIC).

Another form of coupling, especially for the radioelements, can consistin the use of a bifunctional ion chelator.

Among these chelates, it is possible to mention the chelates derivedfrom EDTA (ethylenediaminetetraacetic acid) or from DTPA(diethylenetriaminepentaacetic acid) which have been developed forbinding metals, especially radioactive metals, and immunoglobulins.Thus, DTPA and its derivatives can be substituted by different groups onthe carbon chain in order to increase the stability and the rigidity ofthe ligand-metal complex (Krejcarek et al., (1977); Brechbiel et al.,(1991); Gansow (1991); U.S. Pat. No. 4,831,175).

For example diethylenetriaminepentaacetic acid (DTPA) and itsderivatives, which have been widely used in medicine and in biology fora long time either in their free form, or in the form of a complex witha metallic ion, have the remarkable characteristic of forming stablechelates with metallic ions and of being coupled with proteins oftherapeutic or diagnostic interest such as antibodies for thedevelopment of radioimmunoconjugates in cancer therapy (Meases et al.,(1984); Gansow et al., (1990)).

Likewise preferably, said at least one antibody forming said conjugateaccording to the invention is chosen from its functional fragments,especially the fragments amputated of their Fc component such as thescFv fragments.

The present invention moreover comprises the use of the compositionaccording to the invention for the preparation of a medicament.

More particularly, according to another embodiment, the inventionconcerns the use of an antibody, or one of its functional fragments,and/or of a composition for the preparation of a medicament intended forthe prevention or for the treatment of an illness induced by anoverexpression and/or an abnormal activation of the IGF-IR and/orhybrid-R and/or EGFR, and/or connected with a hyperactivation of thetransduction pathway of the signal mediated by the interaction of theIGF1 or IGF2 with IGF-IR and/or IGF1, IGF2 or insulin with hybrid-Rand/or of EGF with EGFR and/or HER2/neu.

In the present specification, by the object of the invention “use of aproduct or a composition for the preparation of a medicament intendedfor the prevention or for the treatment of a disease”, it is alsocomprised “a method of preventing or treatment of such diseasecomprising the administration of said product or composition in apatient in need of such treatment”

Preferably, said use according to the invention is characterized in thatthe administration of said medicament does not induce or induces onlyslightly secondary effects connected with inhibition of the insulinreceptor IR, that is to say inhibition of the interaction of the IRreceptor with its natural ligands due to the presence of saidmedicament, especially by a competitive inhibition connected with theattachment of said medicament to the IR.

The present invention moreover comprises the use of an antibody, or oneof its functional fragments, preferably humanized, and/or of acomposition according to the invention for the preparation of amedicament intended to inhibit the transformation of normal cells intocells with tumoral character, preferably IGF-dependent, especially IGF1-and/or IGF2-dependent and/or EGF-dependent and/or HER2/neu-dependentcells.

The present invention likewise relates to the use of an antibody, or oneof its functional fragments, preferably humanized, and/or of acomposition according to the invention for the preparation of amedicament intended to inhibit the growth and/or the proliferation oftumor cells, preferably IGF-dependent, especially IGF1- and/orIGF2-dependent and/or EGF-dependent and/or estrogen-dependent, and/orHER2/neu-dependent cells.

In a general manner, a subject of the present invention is the use of anantibody, or one of its functional fragments, preferably humanized,and/or of a composition according to the invention, for the preparationof a medicament intended for the prevention or for the treatment ofcancer preferably expressing, or with an abnormal activation of, IGF-IRand/or hybrid-R and/or EGFR, and/or of cancer preferably having ahyperactivation of the transduction pathway of the signal mediated bythe interaction between IGF-IR and its native ligans, preferably IGF1and IGF2, such as, for example, the overexpression of IRS1 and/orbetween the hybrid-R and its native ligands, preferably IGF1, IGF2 andinsulin or between EGFR with its native ligands, preferably EGF.

The subject of the present invention is likewise the use of an antibody,or one of its functional fragments, preferably humanized, and/or of acomposition according to the invention, for the preparation of amedicament intended for the prevention or for the treatment ofpsoriasis, psoriasis whose epidermal hyperproliferation can be connectedwith the expression or the overexpression of IGF-IR and/or hybrid-Rand/or EGFR, and/or with the hyperactivation of the transduction pathwayof the signal mediated by the interaction of IGF-IR and/or hybrid-Rrespectively with their natural ligands (Wraight C. J. et al., Nat.Biotechnol., 2000, 18(5):521-526. Reversal of epidermalhyperproliferation in psoriasis by insulin-like growth factor I receptorantisense oligonucleotides) and/or of EGFR with its natural ligands.

Among the cancers which can be prevented and/or treated, prostatecancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer orcolon cancer or any other cancer overexpressing, or with an abnormalactivation of, IGF-IR and/or hybrid-R is preferred.

According to yet another aspect, a subject of the present invention is amethod of diagnosis, preferably in vitro, of illnesses connected with anoverexpression or an underexpression, preferably an overexpression, ofthe IGF-IR and/or hybrid-R and/or EGFR starting from a biological samplein which the abnormal presence of IGF-IR and/or hybrid-R and/or EGFR issuspected, characterized in that said biological sample is contactedwith an antibody, or one of its functional fragments, according to theinvention, it being possible for said antibody to be, if necessary,labeled.

Preferably, said illnesses connected with the overexpression, or with anabnormal activation of, of the IGF-IR and/or hybrid-R and/or EGFR insaid diagnosis method will be cancers.

Said antibody, or one of its functional fragments, can be present in theform of an immunoconjugate or of a labeled antibody so as to obtain adetectable and/or quantifiable signal.

The antibodies labeled according to the invention or their functionalfragments include, for example, antibodies called immunoconjugates whichcan be conjugated, for example, with enzymes such as peroxidase,alkaline phosphatase, -D-galactosidase, glucose oxydase, glucoseamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malatedehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule suchas biotin, digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can belikewise conjugated to the antibodies or to their functional fragmentsaccording to the invention and especially include fluorescein and itsderivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone etc. In suchconjugates, the antibodies of the invention or their functionalfragments can be prepared by methods known to the person skilled in theart. They can be coupled to the enzymes or to the fluorescent labelsdirectly or by the intermediary of a spacer group or of a linking groupsuch as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraaceticacid (EDTA), diethylene-triaminepentaacetic acid (DPTA), or in thepresence of coupling agents such as those mentioned above for thetherapeutic conjugates. The conjugates containing labels of fluoresceintype can be prepared by reaction with an isothiocyanate.

Other conjugates can likewise include chemoluminescent labels such asluminol and the dioxetanes, bio-luminescent labels such as luciferaseand luciferin, or else radioactive labels such as iodine¹²³, iodine¹²⁵,iodine¹²⁶, iodine¹³³, bromine⁷⁷, technetium^(99m), indium¹¹¹,indium^(113m), gallium⁶⁷, gallium⁶⁸, ruthenium⁹⁵, ruthenium⁹⁷,ruthenium¹⁰³, ruthenium¹⁰⁵, mercury¹⁰⁷, mercury²⁰³, rhenium^(99m),rhenium¹⁰¹, rhenium¹⁰⁵, scandium⁴⁷, tellurium^(121m), tellurium^(122m),tellurium^(125m), thulium¹⁶⁵, thulium¹⁶⁷, thulium¹⁶⁸, fluorine¹⁸,yttrium¹⁹⁹, iodine¹³¹. The methods known to the person skilled in theart existing for coupling the therapeutic radioisotopes to theantibodies either directly or via a chelating agent such as EDTA, DTPAmentioned above can be used for the radioelements which can be used indiagnosis. It is likewise possible to mention labeling with Na[I¹²⁵] bythe chloramine T method [Hunter W. M. and Greenwood F. C. (1962) Nature194:495] or else with technetium^(99m) by the technique of Crockford etal., (U.S. Pat. No. 4,424,200) or attached via DTPA as described byHnatowich (U.S. Pat. No. 4,479,930).

Thus, the antibodies, or their functional fragments, according to theinvention can be employed in a process for the detection and/or thequantification of an overexpression or of an underexpression, preferablyan overexpression, or with an abnormal activation of, of the IGF-IRand/or hybrid-R and/or EGFR in a biological sample, characterized inthat it comprises the following steps:

a) the contacting of the biological sample with an antibody, or one ofits functional fragments, according to the invention; and

b) the demonstration of the IGF-IR and/or hybrid-R and/or EGFR/antibodycomplex possibly formed.

In a particular embodiment, the antibodies, or their functionalfragments, according to the invention, can be employed in a process forthe detection and/or the quantification of the IGF-IR and/or hybrid-Rand/or EGFR in a biological sample, for the monitoring of the efficacyof a prophylactic and/or therapeutic treatment of IGF-, hybrid- and/orEGF-dependent cancer or else of psoriasis.

More generally, the antibodies, or their functional fragments, accordingto the invention can be advantageously employed in any situation wherethe expression, or with an abnormal activation of, of the IGF-IR and/orhybrid-R and/or EGFR must be observed in a qualitative and/orquantitative manner.

Preferably, the biological sample is formed by a biological fluid, suchas serum, whole blood, cells, a tissue sample or biopsies of humanorigin.

Any procedure or conventional test can be employed in order to carry outsuch a detection and/or dosage. Said test can be a competition orsandwich test, or any test known to the person skilled in the artdependent on the formation of an immune complex of antibody-antigentype. Following the applications according to the invention, theantibody or one of its functional fragments can be immobilized orlabeled. This immobilization can be carried out on numerous supportsknown to the person skilled in the art. These supports can especiallyinclude glass, polystyrene, poly-propylene, polyethylene, dextran,nylon, or natural or modified cells. These supports can be eithersoluble or insoluble.

By way of example, a preferred method brings into play immunoenzymaticprocesses according to the ELISA technique, by immunofluorescence, orradio-immunoassay (RIA) technique or equivalent.

Thus, the present invention likewise comprises the kits or setsnecessary for carrying out a method of diagnosis of illnesses induced byan overexpression or an underexpression of the IGF-IR and/or hybrid-Rand/or EGFR or for carrying out a process for the detection and/or thequantification of an overexpression or of an underexpression of theIGF-IR and/or hybrid-Rs and/or EGFR in a biological sample, preferablyan overexpression, or with an abnormal activation of, of said receptor,characterized in that said kit or set comprises the following elements:

a) an antibody, or one of its functional fragments, according to theinvention;

b) optionally, the reagents for the formation of the medium favorable tothe immunological reaction;

c) optionally, the reagents allowing the demonstration of IGF-IR and/orhybrid-R and/or EGFR/antibody complexes produced by the immunologicalreaction.

The invention moreover relates to the use of a composition as acombination product according to the invention, for the preparation of amedicament intended for the prevention or for the treatment of cancer,especially cancers for which said cytotoxic agent or said anti-HER2/neuantibody is generally prescribed and, especially, for which cancers thetumor cells express or overexpress the IGF-IR and/or EGFR receptor.

A subject of the invention is likewise the use of an antibody accordingto the invention for the preparation of a medicament intended for thespecific targeting of a biologically active compound to cells expressingor overexpressing the IGF-IR and/or hybrid-R and/or EGFR.

It is intended here by biologically active compound to indicate anycompound capable of modulating, especially of inhibiting, cell activity,in particular their growth, their proliferation, transcription or genetranslation.

A subject of the invention is also an in vivo diagnostic reagentcomprising an antibody according to the invention, or one of itsfunctional fragments, preferably labeled, especially radiolabeled, andits use in medical imaging, in particular for the detection of cancerconnected with the expression or the overexpression by a cell of theIGF-IR and/or hybrid-R and/or EGFR.

The invention likewise relates to a composition as a combination productor to an anti-IGF-IR and/or hybrid-R and/or EGFR/toxin conjugate orradioelement, according to the invention, as a medicament.

Preferably, said composition as a combination product or said conjugateaccording to the invention will be mixed with an excipient and/or apharmaceutically acceptable vehicle.

In the present description, pharmaceutically acceptable vehicle isintended to indicate a compound or a combination of compounds enteringinto a pharmaceutical composition not provoking secondary reactions andwhich allows, for example, facilitation of the administration of theactive compound(s), an increase in its lifespan and/or in its efficacyin the body, an increase in its solubility in solution or else animprovement in its conservation. These pharmaceutically acceptablevehicles are well known and will be adapted by the person skilled in theart as a function of the nature and of the mode of administration of theactive compound(s) chosen.

Preferably, these compounds will be administered by the systemic route,in particular by the intravenous route, by the intramuscular,intradermal, intraperitoneal or subcutaneous route, or by the oralroute. In a more preferred manner, the composition comprising theantibodies according to the invention will be administered severaltimes, in a sequential manner.

Their modes of administration, dosages and optimum pharmaceutical formscan be determined according to the criteria generally taken into accountin the establishment of a treatment adapted to a patient such as, forexample, the age or the body weight of the patient, the seriousness ofhis/her general condition, the tolerance to the treatment and thesecondary effects noted.

The antibody, or fragments thereof, could be use alone or in associationwith another antibody able to target another growth factor implied inthe proliferation or dissemination of tumoral cells. It could also beused in association with a chemotherapeutic agent or another tyrosinekinase inhibitor in co-administration or in the form of animmuno-conjugate, said agent being chemical, biological and/or natural.Fragments of said antibody could also be use in bispecific antibodiesobtained by recombinant mechanisms or biochemical coupling, and thenassociating the specificity of the above described antibody with thespecificity of other antibodies able to recognize other receptorsinvolved in the proliferation, the angiogenese or any other mechanismsinvolved in the tumoral development.

Particular aspect of the present invention: Cytotoxic and/or cytostaticactive agent coupled to an addressing system, particularly to theantibodies 7C10, C7C10 or h7C10, or fragment thereof, according to thepresent invention capable of binding specifically to the humaninsulin-like growth factor-1 receptor IGF-IR and hybrid-R.

The present invention relates also to novel compounds comprising acytotoxic and/or cytostatic active agent coupled to an addressingsystem. More particularly, the present invention relates to a compoundcomprising a Vinca alkaloid coupled to an antibody capable of bindingspecifically to the human insulin-like growth factor-1 receptor IGF-IRand/or capable of specifically inhibiting the tyrosine kinase activityof said IGF-IR receptor, in particular a monoclonal antibody of murine,chimeric, primatized, humanized and human origin. The invention alsorelates to the mode of coupling of the elements of said compound andalso comprises the use of these compounds as a medicinal product for theprophylactic and/or therapeutic treatment of cancer, more particularlyof cancers overexpressing IGF-IR, or of any pathological conditionassociated with overexpression, or with an abnormal activation of, ofsaid receptor.

Currently, along with surgery and radiotherapy, chemotherapy representsone of the most effective means of combating cancer. Many cytotoxicand/or cytostatic agents have been isolated or synthesized and make itpossible to destroy or reduce, if not definitively, at leastsignificantly, the tumor cells. However, the toxic activity of theseagents is not limited to tumor cells, and the non-tumor cells are alsoeffected and can be destroyed. More particularly, side effects areobserved on rapidly renewing cells, such as haematopoietic cells orcells of the epithelium, in particular of the mucous membranes. By wayof illustration, the cells of the gastrointestinal tract are largelyeffected by the use of cytotoxic agents.

One of the aims of the present invention is also to be able to provide acompound which makes it possible to limit the side effects on normalcells while at the same time conserving a high cytotoxicity on tumorcells.

According to an original approach, the applicant, rather than developingnew molecules, has sought to overcome the problem of toxicity of knownmolecules by limiting to tumor cells the access of said molecules. To dothis, the applicant has developed an antibody-type addressing system fortargeting only tumor cells.

One of the advantages of this approach is to be able to use knowncytotoxic agents which are well defined in pharmacological andpharmacokinetic terms. In addition, it is then possible to use strongcytotoxic agents which until now have been neglected in favor ofcytotoxic agents which are less strong but which have a bettertherapeutic index (and therefore exhibit fewer side effects).

Another advantage lies in the use of an antibody, i.e., of a product ofbiological origin which does not add any toxicity to that of thecytotoxic agent. In addition, as will be subsequently developed, thechoice of the antibody makes it possible to accumulate with the actionof the cytotoxic agent its own biological activity.

The applicant has demonstrated that the use of a Vinca alkaloid coupledto an addressing device is of value in chemotherapy.

According to a first aspect, a subject of the present invention is acompound comprising at least one molecule of active agent coupled to anaddressing system, said at least one molecule of active agent being astrong cytotoxic and/or cytostatic compound chosen from Vinca alkaloids,and said addressing system being a polyclonal or monoclonal antibody,which may be bispecific, or a functional fragment thereof, capable oftargeting, preferably specifically, tumor cells.

An advantage of a compound according to the invention is that the activeagent is directly brought to the target cells by the antibody and,besides the fact that it does not degrade the other cells, itsbiological activity is not decreased.

One of the advantages associated with using antibodies as an addressingsystem is that it is possible to couple several active agents to them,thus increasing the efficacy of the compound. Specifically, since thecompound is brought directly to the target cells, the fact that thereare several active agents will not lead to an increase in side effects,but only to an increase in the desired in situ effect on the tumorcells.

By way of non-limiting examples of targeting antibodies which can beused according to the invention, mention may be made, without anylimitation, of the CeaVac antibodies directed against colorectal tumorcells, and the Y Theragyn/pemtumomab and OvaRex antibodies directedagainst ovarian tumor cells.

The present invention relates to a compound as described above, whichcomprises from 1 to 50 molecules of active agent, preferably from 1 to10, and better still from 1 to 6.The choice of the number of moleculesof active agent depends, inter alia, on the molecular weight of each ofthe elements. For example, by way of indication, for an antibody of IgG1type with a molecular weight of 150 000 Da, it is preferred to couplefrom 4 to 6 molecules of vinblastine with a molecular weight of 900 Da(Petersen et al., Cancer Res., 1991, 51:2286). If the antibody isconjugated with too large an amount of cytotoxic agents, there is a riskthat said agents will mask the recognition site for the antigen anddecrease its activity.

In practice, the compound which is the subject of the invention is usedas a medicinal product, and more particularly as an medicinal productintended for the treatment of cancer.

The present invention differs from the prior art not only in the sensethat the choice of the antibody is aimed at targeting tumor cells asdescribed above, but also in that said antibody exhibits an intrinsicactivity on the tumor cells.

According to another embodiment of the invention, the compound asdescribed above is also capable of inhibiting tumor cell proliferationand/or apoptotic function restoration by blocking transduction signals,the progression of cells in the cell cycle and/or membrane-boundreceptor availability (phenomena of internalization and of degradationof said receptor), or of reverting an apoptosis-resistant phenotype inthe case of an antibody directed against the IGF-IR, insofar as it iswidely described that overexpression of this receptor confers on tumorcells a means of withstanding apoptosis and in particular apoptosisinduced by chemotherapy compounds (Beech D. J. et al., Oncology reports,2001, 8:325-329; Grothe A. et al., J. Cancer Res. clin Oncol., 1999,125:166-173). Another mechanism of action of the compound as describedabove may be associated with the Fc portion of the antibody, if a wholeantibody is used, and may consist of the setting up of effectormechanisms such as ADCC (antibody-dependent cellular cytotoxicity) andCDC (complement-dependent cytotoxicity).

By way of non-limiting example of antibodies, mention may be made ofAvastin/Bevacizumab which acts on colorectal cancers by interfering withtumor angiogenesis, Rituxan/rituximab, the activity of which is mainlyrelated to the effector functions of the molecule, and in particularADCC, and also Herceptin/trastuzumab which acts by inhibition of signaltransduction and inhibition of cell progression in the cell cycle, andalso, in large part, by initiating ADCC mechanisms.

Vinca alkaloids correspond to the family of natural compounds of whichvinblastine, vincristine, anhydrovinblastine and leurosine, which arepresent in considerable amounts in plants, are demonstrative examples.

The term “Vinca alkaloids” should also be understood to mean all thederivatives present in small amounts, such as deoxyvinblastine orleurosidine, taken by way of non-limiting examples. It should also beunderstood to mean derivatives of natural structure but which areobtained by synthesis, such as, without any limitation,anhydrovinblastine.

The term “Vinca alkaloid” should also be understood to mean all thecompounds derived from these natural compounds by chemical orbiochemical modification in one or more steps. These modifications mayaffect the “vindoline” component or the “velbanamine” component or bothcomponents simultaneously. The Vinca alkaloids, as such, are known tothose skilled in the art (Antitumor Bisindole Alkaloids fromCatharanthus roseus (L.)). The Alkaloids, Brossi A. et al., M. Ed.Academic Press Inc. San Diego, vol. 37, 1990; Jacquesy J. C. et al.,Biomedical Chemistry: Applying Chemical Principles to the Understandingand Treatment of Disease, edited by Torrence, P. F., John Wiley and SonsInc.: New York, 2000, pp. 227-246; Fahy J. et al., J. Current Pharm.Des., 2001, 7:1181-97; Duflos A. et al., Novel Aspects of Natural andModified Vinca Alkaloids, Curr. Med. Chem.—Anti-Cancer Agents, 2002,2:55-70).

The preferred derivatives according to the present invention are thosewhich exhibit a pharmacological advantage established by virtue ofcytotoxicity assays or activity assays on certain specific targets, suchas tubulin, or which have demonstrated advantages in in vivo tests onanimals. Among these compounds, mention may be made of the derivativescurrently used in anticancer chemotherapy: vinblastine, vincristine,vindesine and vinorelbine, and also the derivatives which havedemonstrated an advantage in clinical studies, such as vinepidine,vinfosiltine, vinzolidine and vinflunine.

The invention is therefore partly based on the choice of an originalcytotoxic agent without any bias from the prior art.

More particularly, a subject of the present invention is a compound asdescribed above, in which said Vinca alkaloid is selected fromvinblastine, deoxyvinblastine, deoxyleurosidine, vincristine, vindesine,vinorelbine, vinepidine, vinfosiltine, vinzolidine and vinflunine.

The subject of the invention has, more specifically, been demonstratedand exemplified using deoxyvinblastine and its 4′-S isomer, commonlyknown as deoxyleurosidine.

The structure of each of these two compounds has been described for manyyears, but their pharmacological activity is considered to be moderateor weak (Neuss N. et al., Tetrahedron Letters, 1968, No. 7, pp 783-7;U.S. Pat. No. 4,143,041, Eli Lilly and Company, Filed Nov. 25, 1977; andrecently, Kuehne M. E. et al., J. Org. Chem., 1989, 54, 14:3407-20;Kuehne M. E., Org. Biomol. Chem., 2003 1:2120-36). Their real advantageas a compound with unquestionable antitumour pharmacological activityhas never been described and demonstrated by in vivo experiments onmurine tumor models.

The present invention therefore relates to a compound as describedabove, in which said Vinca alkaloid is (4′-R) deoxyvinblastine and/or(4′-S) deoxyleurosidine.

The greater activity of these two derivatives has been demonstratedagainst P388 murine leukemia grafted intravenously on day 0.The compoundis administered intraperitoneally in a single dose on day 1.The protocolfor this test is described by Kruczynski A. et al., Cancer Chemotherapyand Pharmacology, 1998, volume 41, pages 437 to 447.

Conventionally, the in vivo activity of cytotoxic compounds is expressedby the T/C at a dose expressed in mg per kg. The T/C corresponds to theratio, multiplied by 100, of the median of the survival time of thetreated animals to the median of the survival time of the controlanimals.

By way of example, for cytotoxic agents used to date, the maximumactivity of vinblastine sulfate is expressed at the dose of 5 mg/kg,with T/C=143.The maximum activity of vincristine sulfate is expressed atthe doses of 1.25 and 2.5 mg/kg, with T/C=143 in both cases.

Unexpectedly, the maximum activity of deoxyvinblastine ditartrate isexpressed at the dose of 20 mg/kg, with T/C=214 and the maximum activityof deoxyleurosidine ditartrate is expressed at the dose 2.5 mg/kg, withT/C=200.

In view of these results, the present invention therefore relates to theuse of (4′-R) deoxyvinblastine and/or (4′-S) deoxyleurosidine,collectively referred to as deoxyvinblastine in the remainder of thedescription, for treating cancer.

According to a preferred form, as described above, the present inventionenvisages the coupling of deoxyvinblastine to a compound of themonoclonal or polyclonal, preferably monoclonal, antibody type.

More particularly, as will subsequently be described, a preferredantibody making up the compound which is the subject of the presentinvention is a monoclonal or polyclonal, preferably monoclonal, antibodywhich will recognize the IGF-IR specifically and with high affinity, andwhich will have the ability to inhibit the growth of tumors, moreparticularly of tumors expressing the IGF-IR.

The cytoplasmic protein tyrosine kinases are activated by binding of theligand to the extracellular domain of the receptor. Activation of thekinases leads, in turn, to stimulation of various intracellularsubstrates, including IRS-1, ISR-2, Shc and Grb 10 (Peruzzi F. et al.,J. Cancer Res. Clin. Oncol., 125:166-173, 1999). The two majorsubstrates for the IGF-IR are IRS and Shc, which mediate, by activationof many downstream effectors, most of growth and differentiation effectsassociated with the binding of IGFs to this receptor. Substrateavailability can, consequently, dictate the final biological effectassociated with activation of the IGF-IR. When IRS-1 predominates, thecells tend to proliferate and to transform. When Shc dominates, thecells tend to differentiate (Valentinis B. et al., J. Biol. Chem.,274:12423-12430, 1999). It appears that the pathway mainly implicatedfor the effects of protection against apoptosis is thephosphatidylinositol 3-kinases (PI 3-kinases) pathway (Prisco M. et al.,Horm. Metab. Res., 31:80-89, 1999; Peruzzi F. et al., J. Cancer Res.Clin. Oncol., 125:166-173, 1999).

According to a preferred embodiment, a subject of the present inventionis a compound as described above (cytotoxic and/or cytostatic activeagent coupled to an addressing system), comprising an antibody capableof recognizing the IGF-IR specifically and with high affinity. Thisantibody will interact little or not at all with the insulin receptorIR. Its binding should inhibit, in vitro, the growth of tumorsexpressing the IGF-IR by interacting mainly with the signal transductionpathways activated during IGF1/IGF-IR and IGF2/IGF-IR interactions. Thisantibody should be active in vivo on all tumor types expressing theIGF-IR, including oestrogen-dependent breast tumors and prostate tumors,which is not the case for the anti-IGF-IR monoclonal antibodies(referred to as MAb or MAB) currently available. In fact, αIR3, which isa reference in the IGF-IR field, completely inhibits the growth ofoestrogen-dependent breast tumors (MCF-7) in vitro, but has no effect onthe corresponding in vivo model (Artega C. et al., J. Clin. Invest.,84:1418-1423, 1989). Similarly, the scFv-Fc fragment derived from themurine monoclonal 1H7 is only weakly active on the MCF-7 breast tumorand completely inactive on an androgen-independent prostate tumor (Li S.L. et al., Cancer Immunol. Immunother., 49:243-252, 2000).

According to a preferred embodiment, a subject of the present inventionis a compound (cytotoxic and/or cytostatic active agent coupled to anaddressing system) as described above, comprising an antibody, or one ofits functional fragments, said antibody or one of its said fragmentsbeing capable of binding specifically to the human insulin-like growthfactor-I receptor IGF-IR and, where appropriate, capable of inhibitingthe natural binding of the IGF-IR ligands IGF1 and/or IGF2, and/orcapable of specifically inhibiting the tyrosine kinase activity of saidIGF-IR receptor.

Such a compound has a double advantage.

Firstly, it makes it possible, as described above, to bring thecytotoxic agent directly to tumor cells, more particularly tumor cellsoverexpressing, or with an abnormal activation of, the IGF-IR, and thusto decrease the side effects in normal cells.

Secondly, its mode of action is not limited to targeting. The compoundwhich is the subject of the present invention cumulates the action ofthe cytotoxic agent which makes it possible to destroy the tumor cellsand the action of the antibody which will inhibit the growth of tumorcells, preferably of tumor cells expressing, or with an abnormalactivation of, the IGF-IR, by interacting with the signal transductionpathways, and will make it possible to decrease the resistance toapoptosis of cells overexpressing the receptor for IGF-I and,consequently, to improve the activity of chemotherapy drugs, part of themechanism of action of which lies in the induction of apoptosis.

According to a preferred embodiment of the compound (cytotoxic and/orcytostatic active agent coupled to an addressing system) which is thesubject of this particularly object of the present invention, themonoclonal antibody, or one of its functional fragments, is the 7C10, aC7C10 or a h7C10, or fragment thereof, or their derived antibodies, asdescribed in the first part of the present specification directed to theantibodies anti-IGR-IR of the present invention

In this respect, the applicant filed a French patent application FR03/08538 on Jul. 11, 2003 for “Novel antitumour immunoconjugates”. Thecontent of this patent application is incorporated herein by way ofreference.

Immunoliposomes containing such particular cytotoxic and/or cytostaticagents, such as described above, such as the vinca alkaloids, and ofaddressing them to tumor cells by means of antibodies or of antibodyfragments attached to their surface are comprised in the presentinvention.

Method of treatment of cancer, particularly the preferred cancers citedabove, comprising the administration of the present immunoliposomesforms also part of the present invention.

The antibodies or antibody fragments used are directed against antigensoverexpressed at the surface of tumor cells and/or surface antigens theexpression of which is restricted to tumor cells. They are preferablydirected against tyrosine kinase receptors, and more particularlyagainst the receptors for IGF-I, EGF or else VEGF. A preferred antibodyis a monoclonal or polyclonal, preferably monoclonal, or even humanized,antibody which will recognize the IGF-IR specifically and with highaffinity. Even more preferably, this antibody consists of the antibodyanti-IGR-IR which is the subject of the present invention described inthe first part of the specification.

According to another embodiment of the compound (cytotoxic and/orcytostatic active agent coupled to an addressing system) which is asubject of the present invention, the monoclonal antibody as describedabove is also capable of binding specifically to the human epidermalgrowth factor receptor, EGFR, and/or capable of specifically inhibitingthe tyrosine kinase activity of said EGFR.

According to a preferred aspect of this embodiment of thecompound(cytotoxic and/or cytostatic active agent coupled to anaddressing system), the coupled monoclonal antibody consists of abispecific antibody comprising a second unit which specifically inhibitsthe binding of EGF to the EGFR and/or which specifically inhibits thetyrosine kinase activity of said EGFR.

In a preferred embodiment of the invention, the bispecific antibodywhich can be used here for cytotoxic and/or cytostatic active agentcoupled to an addressing system according to this invention is those asdescribed in the first part of the present specification related tobispecific antibodies of the invention.

Another aspect of the invention concerns the mode of coupling betweenthe antibody and the cytotoxic agent. Whatever the nature of thecoupling, which may be direct or indirect, stable or labile, it shouldin no way impair the respective biological functions of the antibody andof the cytotoxic agent. It is clearly understood that any couplingsatisfying this characteristic, and known to those skilled in the art,is included in the scope of the present patent application. In addition,the coupling, and more particularly the linkage used, must allow releaseof the deoxyvinblastine, in the 4-deacetylated or 3-acid, or4-deacetylated and 3-acid, form, or in the form of one of these formscarrying all or part of said linkage used, in the target cells.

According to a preferred embodiment, the coupling is chemical coupling.More particularly, said chemical coupling is composed of an anchorage onthe Vinca alkaloid, an anchorage on the antibody and a linkageconnecting these two anchorages.

The term “linkage” should be understood to mean any structure capable ofproviding a bond of whatever possible nature between the two elements ofthe compound, namely a chemical molecule and an antibody.

In terms of the anchorage on the Vinca alkaloid, several possibilitiesare envisaged. Mention may, for example, be made of an anchorage on thealcohol function in the 4-position after deacetylation of the 4-acetoxygroup of said Vinca alkaloid.

In another embodiment, the anchorage on the Vinca alkaloid is effectedon the acid function in the 3-position after deacetylation of the4-acetoxy group and demethylation of the ester function in the3-position of said Vinca alkaloid.

According to yet another embodiment of the invention, the anchorage onthe Vinca alkaloid is effected on the acid function in the 3-positiondirectly by reaction on the ester function in the 3-position of saidVinca alkaloid.

According to yet another embodiment of the invention, the anchorage onthe Vinca alkaloid is effected via an ester or thioester function on thehydroxyl function in the 3-position.

An additional embodiment consists in effecting the anchorage on theVinca alkaloid via an amide function or an ester function or a hydrazidefunction on the acid function in the 4-position.

As regards the anchorage on the antibody, it should in no way denaturethe antibody, so as not to decrease its ability to recognize andinteract with the tumor cells.

To do this, it is preferable for the anchorage on the antibody to beeffected on the oligosaccharides, the lysines and/or the aspartic acidand glutamic acid residues.

The Vinca alkaloid may also be coupled on the carboxylic functions ofthe antibody, carried by the aspartic acid and glutamic acid residues ofthe antibody. For example, an amine, hydrazide or hydrazine derivativeof the Vinca alkaloid will be coupled on these residues in the presenceof a compound of carbodiimide type, such asN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (or EDAC).

In practice, it is even more preferable to effect the anchorage on theoligosaccharides present on the antibody. Specifically, there are nooligosaccharides in the recognition sites of the antibody and, as aresult, there is no risk of impairing the recognition/biologicalactivity capacities of said antibody. According to a preferredembodiment of the invention, the anchorage is effected on theoligosaccharides present on the asparagines (Asn) which are followed bya consensus sequence consisting of an amino acid and a serine or athreonine. For example, without any limitation, a preferred anchorage onthe IgG1 antibody used in the invention is on Asn297.

A combined anchorage, i.e., an anchorage on oligosaccharides, lyinesand/or aspartic acid and glutamic acid, is also covered.

An additional embodiment consists in greatly increasing the density ofthe Vinca alkaloid in order to attain 10 to 50 mol per mole of antibody.Mention may be made of the coupling of a hemisuccinate derivative of theVinca alkaloid on a lysine polymer (Poly-L-Lys or Poly-D-Lys). Theconjugate thus obtained is then coupled on the oligosaccharides of theantibody, oxidized beforehand with meta-periodate.

In another embodiment, a hydrazide derivative of the Vinca alkaloid maybe coupled on a dextran oxidized beforehand with meta-periodate. Theconjugate obtained is then coupled to the antibody via the lysineresidues.

According to yet another embodiment, a hemisuccinate derivative of theVinca alkaloid may be coupled on a dextran activated beforehand bycontrolled oxidation with meta-periodate and then substituted with acompound of diamide type. The conjugate obtained is then coupled on thelysine residues of the antibody.

According to a preferred embodiment of the invention, the anchorage onthe antibody is effected by reaction of an amine function, a hydrazinefunction, a hydrazide function or an acid function which has beenactivated.

More particularly, the anchorage on the antibody is effected by reactionof an epoxide function or of a disulfide function, a sulfide function oran acid function which has been activated, with a nitrogen-containingresidue or with a hydroxyl residue or with a thiol residue of saidantibody.

Mention may also be made, in a nonlimiting manner, of other linkageswhich may also be used to covalently attach the Vinca alkaloids to theantibodies or to their functional fragments (Garnett et al., Adv. DrugDeliv. Rev., 2001, 171-216), such as aldehydes which make it possible toform Schiff bases, which can then be stabilized by reduction with sodiumborohydride or cyanoborohydride; disulfides which have the advantage ofbeing able to release the Vinca alkaloids inside the tumor cell byvirtue of the intracytoplasmic reducing environment; more stablethioethers; more labile thioesters; linkages which are labile in acidicmedium, which have the advantage of allowing release of the cytotoxicagent in the tumor, which is generally more acid, or during the passagefrom the endosome (pH 6.0 6.8) to the lysosome (pH 4.5 5.5), or elseenzyme-degradable linkages which have the advantage of being stable inthe serum and of releasing the cytotoxic agent in the intracellularmedium of the tumor cell.

Mention may also be made of peptide sequences of the Ala Leu type, whichcan be cleaved by lysosomal hydrolases (Masquelier et al., J. Med.Chem., 1980, 23:1166-1170) or else linkages of the hydrazone type, suchas those used in the gemtuzumab ozogamicin immunoconjugate used in thetreatment of certain types of leukemia and sold under the name Mylotarg(Hamann et al., Bioconjugate Chem., 2002, 13:47).

As described above, a preferred form of the invention uses a linkagewhich allows release of the deoxyvinblastine in the tumor cells.

A first means for achieving this consists in using a linkage connectingthe two anchorages which consists of a peptide chain. In fact, such apeptide linkage will be degraded/hydrolyzed in the target cells by theenzymes of the endosomes and of the lysosomes.

According to another embodiment of the invention, the linkage connectingthe two anchorages consists of a linear or branched carbon-based chain.In the latter case, it is envisaged that one or more aromatic, ethylenicor acetylenic groups and also one or more ketone, amide, ester,hydrazide, hydrazone, amine, ether, sulfide or disulfide groups areincluded in the carbon chain in a distinct or combined manner. Forexample, in the case of an attachment via a disulfide bridge, it is thereducing medium which will allow cleavage of the linkage and release ofthe deoxyvinblastine.

In all cases, only the linkage is destroyed in order to release theactive principle, said active principle and the antibody themselvesremaining intact.

According to yet another embodiment, there is no linkage, but the Vincaalkaloid is coupled directly with a nitrogen-containing residue or witha hydroxyl residue or with a thiol residue of the antibody.

The advantage of such a direct coupling lies in the absence of anchoragelinkage and, consequently, in the absence of an immune reaction by thepatient against this linkage. The appearance of anti-linkage antibodiessecreted by the body in response to the intrusion of said linkage isthus, for example, avoided.

More particularly, the compound according to the invention ischaracterized in that the acid function in the 4-position of the Vincaalkaloid is coupled, via a hydrazide function, with an aldehyde residueof the antibody, generated beforehand.

The invention also relates to a pharmaceutical composition comprising,as active principle, a compound consisting of a Vinca alkaloid coupledto an antibody, or one of its functional fragments, according to theinvention, to which a pharmaceutically acceptable excipient and/orvehicle is preferably added.

The present invention also comprises the use of the compound accordingto the invention for preparing a medicinal product.

More particularly, according to another embodiment, the inventionrelates to the use of a compound as described above and/or of acomposition comprising such a compound, for preparing a medicinalproduct intended for the prevention or treatment of cancers, inparticular cancers induced by overexpression and/or activation, or withan abnormal activation of, of the IGF-IR and/or EGFR receptor which isabnormal, and/or associated with hyperactivation of the signaltransduction pathway mediated by the interaction of IGF1 or IGF2 withIGF-IR and/or of EGF with EGFR.

Among the cancers which may be prevented and/or treated, prostatecancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer orcolon cancer, or any other cancer overexpressing IGF-IR, is preferred.

According another aspect, the invention concerns a humanized anti-IGF-IRantibody, or antigen-binding fragment thereof, wherein said anti-IGF-IRantibody, or antigen-binding fragment, comprises at least onecomplementary determining region of non-human origin and at least oneframework region having at least one human residue, wherein saidantibody is characterized as:

a) binding IGF-IR but not IR alone; and/or

b) inhibiting the binding between a human IGF-IR and its native ligand,preferably designated herein as IGF1 and/or IGF2, with an inhibitionconstant and/or IC₅₀ of less than 100 nM, preferably less than 50 nM;and/or

c) specifically inhibiting the tyrosine kinase activity of said IGF-IR;and/or

d) having a binding affinity of 10 nM or less for said IGF-IR; and/or

e) down-regulating IGF-IR expression; and/or

f) inhibiting in vivo tumor growth;

wherein said antibody comprises a heavy chain amino acid sequencecomprising human FR1, FR2, and FR3 amino acid sequences that correspondto those of a human germline such as, for example, the human Kabatsubgroup I(A) germline, or conservative substitutions or somaticmutations therein, and/or

wherein said antibody comprises a light chain amino acid sequencecomprising human FR1, FR2, and FR3 amino acid sequences that correspondto those of a human germline such as, for example, the human Kabatsubgroup II germline, or conservative substitutions or somatic mutationstherein, and

wherein the FR sequences of the heavy chain are linked with CDR1, CDR2,and CDR3 of sequences derived from a non-human source and comprising theamino acid sequences selected from the group consisting of SEQ ID NOs 8,10 or 12, and/or

wherein the FR sequences of the light chain are linked with CDR1, CDR2,and CDR3 of sequences derived from a non-human source and comprising theamino acid sequences selected from the group consisting of SEQ ID NOs 2,4 or 6.

In a second embodiment, the present invention also deals with ahumanized anti-hybrid-R antibody or antigen-binding fragment thereof,wherein said anti-hybrid-R antibody or antigen-binding fragmentcomprises at least one complementary determining region of non-humanorigin and at least one framework region having at least one humanresidue, wherein said antibody is characterized as:

a) binding hybrid-R but not IR alone; and/or

b) inhibiting the binding between a human hybrid-R and its nativeligand, preferably designated herein as IGF1 and/or IGF2 and/or insulin,with an inhibition constant and/or IC₅₀ of less than 100 nM, preferablyless than 50 nM; and/or

c) specifically inhibiting the tyrosine kinase activity of saidhybrid-R; and/or

d) having a binding affinity of 10 nM or less for said hybrid-R; and/or

e) down-regulating hybrid-R expression; and/or

f) inhibiting in vivo tumor growth;

wherein said antibody comprises a heavy chain amino acid sequencecomprising human FR1, FR2, and FR3 amino acid sequences that correspondto those of a human germline such as, for example, the human Kabatsubgroup I(A) germline, or conservative substitutions or somaticmutations therein,

wherein said antibody comprises a light chain amino acid sequencecomprising human FR1, FR2, and FR3 amino acid sequences that correspondto those of a human germline such as, for example, the human Kabatsubgroup II germline, or conservative substitutions or somatic mutationstherein, and

wherein the FR sequences are linked with CDR1, CDR2, and CDR3 sequencesderived from a non-human source and comprising the amino acid sequencesselected from the group consisting of SEQ ID NOs 8, 10 or 12, and/or

wherein the FR sequences of the light chain are linked with CDR1, CDR2,and CDR3 of sequences derived from a non-human source and comprising theamino acid sequences selected from the group consisting of SEQ ID NOs 2,4 or 6.

Moreover, according a third embodiment of the invention, it is envisageda humanized anti-IGF-IR and hybrid-R antibody or antigen-bindingfragment thereof, wherein said anti-IGF-IR and hybrid-R antibody orantigen-binding fragment comprises at least one complementarydetermining region of non-human origin and at least one framework regionhaving at least one human residue, wherein said antibody ischaracterized as:

a) binding IGF-IR and hybrid-R but not IR alone; and/or

b) inhibiting the binding between a human IGF-IR and its native ligand,preferably designated herein as IGF1 and/or IGF2, with an inhibitionconstant and/or IC₅₀ of less than 100 nM, preferably less than 50 nM,and also a human hybrid-R, and its natural ligand designated herein asIGF1 and/or IGF2 and/or insulin, with an inhibition constant and/or IC₅₀of less than 100 nM, preferably less than 50 nM; and/or

c) specifically inhibiting the tyrosine kinase activity of both saidIGF-IR and hybrid-R; and/or

d) having a binding affinity of 10 nM or less for said hybrid-R and of10 nM or less for said IGF-IR; and/or

e) down-regulating both IGF-IR and hybrid-R expression ; and/or

f) inhibiting in vivo tumor growth;

wherein said antibody comprises a heavy chain amino acid sequencecomprising human FR1, FR2, and FR3 amino acid sequences that correspondto those of a human germline such as, for example, the human Kabatsubgroup I(A) germline, or conservative substitutions or somaticmutations therein, and/or

wherein said antibody comprises a light chain amino acid sequencecomprising human FR1, FR2, and FR3 amino acid sequences that correspondto those of a human germline such as, for example, the human Kabatsubgroup II germline, or conservative substitutions or somatic mutationstherein, and

wherein the FR sequences are linked with CDR1, CDR2, and CDR3 sequencesderived from a non-human source and comprising the amino acid sequencesselected from the group consisting of SEQ ID NOs 8, 10 or 12, and/or

wherein the FR sequences of the light chain are linked with CDR1, CDR2,and CDR3 of sequences derived from a non-human source and comprising theamino acid sequences selected from the group consisting of SEQ ID NOs 2,4 or 6.

The invention also describes a method of modulating IGF-IR activity inIGF-IR-responsive mammalian cells comprising: contacting the cells withan antibody specific for said receptor, wherein said antibody comprisesa light and a heavy chain, said light chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs: 2, 4 or 6 and said heavy chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs 8, 10, or 12.

In a second embodiment, the invention describes a method of modulatinghybrid-R activity in hybrid-R-responsive mammalian cells comprising:contacting the cells with an antibody specific for said receptor,wherein said antibody comprises a light and a heavy chain, said lightchain comprising at least one complementary determining region CDRchosen from the CDRs of sequence SEQ ID NOs: 2, 4 or 6 and said heavychain comprising at least one complementary determining region CDRchosen from the CDRs of sequence SEQ ID NOs 8, 10, or 12.

Then, in a third embodiment, an object of the present invention is amethod of jointly modulating IGF-IR and hybrid-R activities in IGF-IRand hybrid-R-responsive mammalian cells comprising: contacting the cellswith an antibody specific for said receptors, wherein said antibodycomprises a light and a heavy chain, said light chain comprising atleast one complementary determining region CDR chosen from the CDRs ofsequence SEQ ID NOs: 2, 4 or 6 and said heavy chain comprising at leastone complementary determining region CDR chosen from the CDRs ofsequence SEQ ID NOs 8, 10, or 12.

More particularly, the methods above described, and wherein the antibodymodulates IGF-IR activity, is directed against, without limitation,mammalian cells selected from the group consisting of breast cancercells, prostate cancer cells, colorectal cancer cells, lung cancercells, bladder cancer cells, kidney cancer cells, thyroid cancer cells,osteosarcomas cells, pancreatic cancer cells, melanoma and myeloidcells.

The method above described, wherein the antibody modulates hybrid-Ractivity, is directed against, without limitation, mammalian cellsselected from the group consisting of breast cancer cells or thyroidcancer cells.

The invention also deals with method of decreasing IGF-IR activity inIGF-IR-responsive mammalian cells comprising: contacting the cells withan antibody specific for said receptor in an amount sufficient todecrease the activity of IGF-IR, wherein said antibody comprises a lightand a heavy chain, said light chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs: 2, 4 or 6 and said heavy chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs 8, 10, or 12.

In a similar manner than previous method, the invention concerns, in asecond embodiment, a method of decreasing hybrid-R activity inhybrid-R-responsive mammalian cells comprising: contacting the cellswith an antibody specific for said receptor in an amount sufficient todecrease the activity of hybrid-R, wherein said antibody comprises alight and a heavy chain, said light chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs: 2, 4 or 6 and said heavy chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs 8, 10, or 12.

In a third embodiment, it is envisaged a method of jointly decreasingIGF-IR and hybrid-R activities in IGF-IR and hybrid-R-responsivemammalian cells comprising: contacting the cells with an antibodyspecific for said receptors in an amount sufficient to decrease theactivities of IGF-IR and hybrid-R, wherein said antibody comprises alight and a heavy chain, said light chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs: 2, 4 or 6 and said heavy chain comprising at least onecomplementary determining region CDR chosen from the CDRs of sequenceSEQ ID NOs 8, 10, or 12.

The method above mentioned, wherein the antibody decreases IGF-IRactivity, concerns mammalian cells selected from the group consistingof, without limitation, breast cancer cells, prostate cancer cells,colorectal cancer cells, lung cancer cells, bladder cancer cells, kidneycancer cells, thyroid cancer cells, osteosarcomas cells, pancreaticcancer cells, melanoma and myeloid cells.

The method above mentioned, wherein the antibody decreases hybrid-Ractivity, concerns mammalian cells selected from the group consistingof, without limitation, breast cancer cells or thyroid cancer cells. Theinvention also concerns a method of identifying an IGF-IR modulatorcomprising:

a) contacting IGF-IR with an antibody according to the invention able tobind IGF-IR;

b) contacting the complex of (a) with a compound library;

c) identifying a compound which disrupts the complex of (a); and

d) determining whether the compound exhibits agonist or antagonistactivity at the IGF-IR, wherein this activity indicates identificationof an IGF-IR modulator.

In a second embodiment, the invention consists in a method ofidentifying a hybrid-R modulator comprising:

a) contacting hybrid-R with an antibody according to the invention ableto bind hybrid-R;

b) contacting the complex of (a) with a compound library;

c) identifying a compound which disrupts the complex of (a); and

d) determining whether the compound exhibits agonist or antagonistactivity at the hybrid-R, wherein this activity indicates identificationof a hybrid-R modulator.

In a third embodiment, a method of identifying an IGF-IR and hybrid-Rmodulator comprising the following steps is an object of the invention:

a) contacting IGF-IR and hybrid-R with an antibody according to theinvention able to bind IGF-IR and hybrid-R;

b) contacting the complex of (a) with a compound library;

c) identifying a compound which disrupts the complex of (a); and

d) determining whether the compound exhibits agonist or antagonistactivity at the IGF-IR and hybrid-R, wherein this activity indicatesidentification of an IGF-IR and hybrid-R modulator.

The present invention is also describing a method of identifying anIGF-IR modulator comprising:

a) screening a library of peptide sequences, wherein said library ofpeptide sequences bind to IGF-IR, and wherein the library is derivedfrom a peptide sequence comprising at least one sequence selected fromthe group consisting of SEQ ID NOs 2, 4, 6, 8, 10 or 12; and

b) determining whether the amino acid sequence isolated in (a) exhibitsagonist or antagonist activity at IGF-IR-responsive cell selected fromthe group consisting of cell lines displaying IGF-IR, such as, forexample, MCF-7, T47D, BT20, ZR-75-1, MDA-MB-231 for breast cells, PC3and DU145 for prostate cells, A549, A427 and SK-LU-1 for lung cells,HT29, Colo205 and CaCo-2 for colon cells, BC-PAP, FRO and ARO forthyroid cells, SK-OV-3 for ovarian cells, BxPC3, MiaPaCa-2 and LN36 forpancreas cells, SK-ES-1 for renal adrenal cancer sarcoma cells, Daoy,TE-671 and D283 Med for medulloblastoma cells, MM-1S and MM-1R forretinoblastoma multiple myeloma cells and SK-MEL-28 for melanoma cellswherein this activity indicates identification of an IGF-IR modulator.

It is also described a method of identifying a hybrid-R modulatorcomprising:

a) screening a library of peptide sequences, wherein said library ofpeptide sequences bind to hybrid-R, and wherein the library is derivedfrom a peptide sequence comprising at least one sequence selected fromthe group consisting of SEQ ID NOs 2, 4, 6, 8, 10 or 12; and

b) determining whether the amino acid sequence isolated in (a) exhibitsagonist or antagonist activity at hybrid-R-responsive cell selected fromthe group consisting of cell lines displaying hybrid-R, such as, forexample, MDA-MB-231, MDA-MB-157, MDA-MB-468, MDA-MB-453 and ZR-75 forbreast cells and BC-PAP for thyroid cells, wherein this activityindicates identification of an hybrid-R modulator.

Then, in a third embodiment, it is also an object of the invention todevelop a method of identifying an IGF-IR and hybrid-R modulatorcomprising:

a) screening a library of peptide sequences, wherein said library ofpeptide sequences bind to IGF-IR and/or hybrid-R, and wherein thelibrary is derived from a peptide sequence comprising at least onesequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10or 12; and

b) determining whether the amino acid sequence isolated in (a) exhibitsagonist or antagonist activity at IGF-IR and/or hybrid-R-responsive cellselected from the group consisting of cell lines displaying IGF-IRand/or hybrid-R, such as, for example, MCF-7, T47D, BT20, ZR-75-1,MDA-MB-231, MDA-MB-157, MDA-MB-468, MDA-MB-453 and ZR-75 for breastcells, PC3 and DU145 for prostate cells, A549, A427 and SK-LU-1 for lungcells, HT29, Colo205 and CaCo-2 for colon cells, BC-PAP, FRO and ARO forthyroid cells, SK-OV-3 for ovarian cells, BxPC3, MiaPaCa-2 and LN36 forpancreas cells, SK-ES-1 for renal adrenal cancer sarcoma cells, Daoy,TE-671 and D283 Med for medulloblastoma cells, MM-1S and MM-1R forretinoblastoma multiple myeloma cells and SK-MEL-28 for melanoma cells,wherein this activity indicates identification of an IGF-IR and hybrid-Rmodulator.

An object of the invention is a method of treating or preventing amedical condition in a subject, which medical condition is mediated byelevated expression and/or activation of IGF1, comprising administeringa binding composition that specifically binds to IGF-IR comprising atleast a member selected from the group consisting of:

a) a light chain amino acid sequence which comprises CDR-L1 defined bySEQ ID NO: 2, CDR-L2 defined by SEQ ID NO: 4 and CDR-L3 defined by SEQID NO: 6; and/or

b) a heavy chain amino acid sequence which comprises CDR-H1 defined bySEQ ID NO: 8, CDR-H2 defined by SEQ ID NO: 10 and CDR-H3 defined by SEQID NO: 12; to the subject.

In a second embodiment, an object of the invention is a method oftreating or preventing a medical condition in a subject, which medicalcondition is mediated by elevated expression and/or activation ofhybrid-R comprising administering a binding composition thatspecifically binds to hybrid-R comprising at least a member selectedfrom the group consisting of:

a) a light chain amino acid sequence which comprises CDR-L1 defined bySEQ ID NO: 2, CDR-L2 defined by SEQ ID NO: 4 and CDR-L3 defined by SEQID NO: 6; and/or

b) a heavy chain amino acid sequence which comprises CDR-H1 defined bySEQ ID NO: 8, CDR-H2 defined by SEQ ID NO: 10 and CDR-H3 defined by SEQID NO: 12; to the subject.

In a third embodiment, an object of the invention is a method oftreating or preventing a medical condition in a subject, which medicalcondition is mediated by elevated expression and/or activation of IGF-IRand hybrid-R comprising administering a binding composition thatspecifically binds to IGF-IR and hybrid-R comprising at least a memberselected from the group consisting of:

a) a light chain amino acid sequence which comprises CDR-L1 defined bySEQ ID NO: 2, CDR-L2 defined by SEQ ID NO: 4 and CDR-L3 defined by SEQID NO: 6; and/or

b) a heavy chain amino acid sequence which comprises CDR-H1 defined bySEQ ID NO: 8, CDR-H2 defined by SEQ ID NO: 10 and CDR-H3 defined by SEQID NO: 12; to the subject.

Another object of the invention is a method of determining regression,progression or onset of a pathological disorder characterized byincreased expression and/or activation of human IGF-IR relative tonormal comprising incubating a sample obtained from a patient with saiddisorder with a detectable probe that is specific for said human IGF-IRunder conditions favoring formation of a probe/IGF-IR complex, thepresence of which is indicative of the regression, progression or onsetof said pathological disorder in said patient.

In a second embodiment, another object of the invention is a method ofdetermining regression, progression or onset of a pathological disordercharacterized by increased expression and/or activation of humanhybrid-R relative to normal comprising incubating a sample obtained froma patient with said disorder with a detectable probe that is specificfor said human hybrid-R under conditions favoring formation of aprobe/hybrid-R complex, the presence of which is indicative of theregression, progression or onset of said pathological disorder in saidpatient.

In a third embodiment, another aspect of the invention is a method ofdetermining regression, progression or onset of a pathological disordercharacterized by increased expression and/or activation of both humanIGF-IR and hybrid-R relative to normal comprising incubating a sampleobtained from a patient with said disorder with a detectable probe thatis specific for said human IGF-IR and hybrid-R under conditions favoringformation of a probe/hybrid-R and probe. IGF-IR complex, the presence ofwhich is indicative of the regression, progression or onset of saidpathological disorder in said patient.

By references to “normal”, it is intended to normal expression or normalactivation of IGF-IR, hybrid-R or both. Any methods of determiningexpression, or activation, levels known by the man skilled in the artcan be used and is incorporated in the present specification. An exampleof such a method is described in example 42 hereinafter.

In the method above described, the probe is preferably an antibody, saidantibody being preferably labeled with a radioactive label, afluorescent label or an enzyme, and said antibody preferably comprisingthe antibody of the invention as described in the present specification.

Still another aspect of the invention is a method of following progressof a therapeutic regimen designed to alleviate a condition characterizedby abnormal expression and/or activation of human IGF-IR relative tonormal comprising:

a) assaying a sample from a subject to determine level of expressionand/or activation of said IGF-IR at a first time point;

b) administering the antibody of the invention able to bind IGF-IR tosaid subject and assaying level of expression and/or activation of saidIGF-IR at subsequent time points following administration of saidantibody; and

c) comparing said level of said IGF-IR at said subsequent time points tothe level determined in (a) as a determination of effect of saidtherapeutic regimen, wherein a decrease in said level of expressionand/or activation of IGF-IR subsequent to administration of saidanti-IGF-IR antibody indicates a positive progression of the therapeuticregimen designed to alleviate said condition.

In a second embodiment, it is also a aspect of the invention to protecta method of following progress of a therapeutic regimen designed toalleviate a condition characterized by abnormal expression and/oractivation of human hybrid-R relative to normal comprising:

a) assaying a sample from a subject to determine level of expressionand/or activation of said hybrid-R at a first time point;

b) administering the antibody of the invention able to bind hybrid-R tosaid subject and assaying level of expression and/or activation of saidhybrid-R at subsequent time points following administration of saidantibody; and

c) comparing said level of said hybrid-R at said subsequent time pointsto the level determined in (a) as a determination of effect of saidtherapeutic regimen, wherein a decrease in said level of expressionand/or activation of hybrid-R subsequent to administration of saidanti-hybrid-R antibody indicates a positive progression of thetherapeutic regimen designed to alleviate said condition.

In a third aspect, the invention is claiming a method of followingprogress of a therapeutic regimen designed to alleviate a conditioncharacterized by abnormal expression and/or activation of both humanIGF-IR and hybrid-R relative to normal comprising:

a) assaying a sample from a subject to determine level of expressionand/or activation of said IGF-IR and hybrid-R at a first time point;

b) administering the antibody of the invention able to bind both IGF-IRand hybrid-R to said subject and assaying level of expression and/oractivation of said IGF-IR and hybrid-R at subsequent time pointsfollowing administration of said antibody; and

c) comparing said level of said IGF-IR and hybrid-R at said subsequenttime points to the level determined in (a) as a determination of effectof said therapeutic regimen, wherein a decrease in said level ofexpression and/or activation of IGF-IR and hybrid-R subsequent toadministration of said anti-IGF-IR and hybrid-R antibody indicates apositive progression of the therapeutic regimen designed to alleviatesaid condition.

The condition above mentioned comprises i) a tumor that expresses atleast an IGF-IR, preferably breast cancer, prostate cancer, colorectalcancer, lung cancer, bladder cancer, kidney cancer, thyroid cancer,osteosarcomas, pancreatic cancer, melanoma and myeloid cancer ; or ii) atumor that expresses at least a hybrid-R, preferably breast cancer orthyroid cancer.

The methods above described can also include, before the administrationof step b), the step consisting in measuring the concentration of aspecific tumoral antigen in a body sample from the mammal, wherein anelevated concentration of said specific tumoral antigen above areference range for said specific tumoral antigen indicates a increasedrisk for cancer.

Specific timoral antigen are described in example 43 hereinafter.Genetic markers can also be used (see example 43).

Methods above described, preferably methods of treating/preventing amedical condition or methods of following progress of a therapeuticregimen are comprising administering to said mammal the antibody of theinvention in combination with an agent selected from the groupconsisting of a corticosteroid, anti-emetic, cancer vaccine, analgesic,anti-vascular agent, cytokines, non-specific immunostimulant andanti-proliferative agent.

In a preferred embodiment, said anti-emetic agent is selected from thegroup consisting of, without limitation, ondansetron hydrochloride,granisetron hydrochloride, metroclopramide, domperidone, haloperidol,cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine,or tropisetron.

In another preferred embodiment, said analgesic agent is selected fromthe group consisting of, without limitation, ibuprofen, naproxen,choline magnesium trisalicylate, or oxycodone hydrochloride.

In another particularly preferred embodiment, said anti-proliferativeagent is selected from the group consisting of, without limitation,farnesyl protein transferase inhibitors, alpha.v.beta.3 inhibitors,alpha.v.beta.5 inhibitors, p53 inhibitors, VEGF inhibitors, VEGFRinhibitors, EGFR inhibitors and Her2neu inhibitors or heterodimersthereof, and PDGFR inhibitors.

Methods above described, preferably methods of treating/preventing amedical condition or methods of following progress of a therapeuticregimen are characterized in that said antibody comprises a heavy chaincomprising the amino acid sequences of CDR-1, CDR-2, and CDR-3 whereinsaid heavy chain CDR's are selected from the group consisting of SEQ IDNOs. 8, 10 or 12, and a light chain comprising the amino acid sequencesof CDR-1, CDR-2, and CDR-3, wherein said light chain CDR's are selectedfrom the group consisting of SEQ ID NOs. 2, 4 or 6, or sequences havingchanges from said CDR sequences selected from the group consisting ofconservative changes, wherein said conservative changes are selectedfrom the group consisting of replacement of nonpolar residues by othernonpolar residues, replacement of polar charged residues by other polaruncharged residues, replacement of polar charged residues by other polarcharged residues, and substitution of structurally similar residues; andnon-conservative substitutions, wherein said non-conservativesubstitutions are selected from the group consisting of substitution ofpolar charged residue for polar uncharged residues and substitution ofnon-polar residues for polar residues, additions and deletions.

In another aspect of the invention, it is claimed a pharmaceuticalcomposition for the treatment or prevention of a disorder in a mammalcomprising an amount of a human anti-IGF-IR antibody that is effectivein treating said disorder and a pharmaceutically acceptable carrier,wherein said disorder is selected from the group consisting of breastcancer, prostate cancer cells, colorectal cancer cells, lung cancercells, bladder cancer cells, kidney cancer cells, thyroid cancer cells,osteosarcomas cells, pancreatic cancer cells, and myeloid cells, whereinsaid antibody comprises the antibody of the invention above describedand able to bind IGF-IR.

Another embodiment is a pharmaceutical composition for the treatment orprevention of a disorder in a mammal comprising an amount of a humananti-hybrid-R antibody that is effective in treating said disorder and apharmaceutically acceptable carrier, wherein said disorder is selectedfrom the group consisting of breast cancer and thyroid cancer, whereinsaid antibody comprises the antibody of the invention able to bindhybrid-R.

Another embodiment is also a pharmaceutical composition for thetreatment or prevention of a disorder in a mammal comprising an amountof a human anti-IGF-IR and hybrid-R antibody that is effective intreating said disorder and a pharmaceutically acceptable carrier,wherein said disorder is selected from the group consisting of breastcancer and thyroid cancer, wherein said antibody comprises the antibodyof the invention able to bind IGF-IR and hybrid-R.

Said pharmaceutical composition(s) is further comprising an amount ofanti-emetic, cancer vaccine, analgesic, anti-vascular agent, andanti-proliferative agent that, in combination with said antibody, iseffective in treating said disorder.

The invention concerns, in another aspect, a method of detecting abinding partner for the antibody of the invention able to bind IGF-IR ina sample, the method comprising:

a) incubating the antibody of the invention able to bind IGF-IR with abiological sample obtained from a patient with a cell proliferativedisorder characterized by abnormal level of expression and/or activationof human IGF-IR under conditions sufficient to allow specific binding ofthe antibody to its binding partner, and

b) detecting specific binding, wherein specific binding indicates thepresence of a binding partner in the sample.

A second embodiment is a method of detecting a binding partner for theantibody of the invention able to bind hybrid-R in a sample, the methodcomprising:

a) incubating the antibody of the invention able to bind hybrid-R with abiological sample obtained from a patient with a cell proliferativedisorder characterized by abnormal level of expression and/or activationof human hybrid-R under conditions sufficient to allow specific bindingof the antibody to its binding partner, and

b) detecting specific binding, wherein specific binding indicates thepresence of a binding partner in the sample.

A third embodiment of the invention is a method of detecting bindingpartners for the antibody of the invention able to bind IGF-IR andhybrid-R in a sample, the method comprising:

a) incubating the antibody of the invention able to bind IGF-IR andhybrid-R with a biological sample obtained from a patient with a cellproliferative disorder characterized by abnormal level of expressionand/or activation of both human IGF-IR and hybrid-R under conditionssufficient to allow specific binding of the antibody to its bindingpartner, and

b) detecting specific binding, wherein specific binding indicates thepresence of a binding partner in the sample.

The invention also deals with a method of purifying a hybrid receptorfrom a sample, the method comprising:

a) incubating the antibody of the invention able to bind hybrid-R with asample under conditions to allow specific binding of the antibody andthe receptor, and

b) separating the antibody from the sample and obtaining the purifiedreceptor.

A second embodiment is a method of purifying a IGF-IR from a sample, themethod comprising:

a) incubating the antibody of the invention able to bind IGF-IR with asample under conditions to allow specific binding of the antibody andthe receptor, and

b) separating the antibody from the sample and obtaining the purifiedreceptor.

At least, it is also an object of the invention to protect the use ofthe biological data obtained according to one of the methods of theinvention for the determination and/or modification of a therapeutictreatment or regimen.

Other characteristics and advantages of the invention appear in thecontinuation of the description with the examples and the figures whoselegends are represented below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of IGF-IR.

FIG. 2: Scheme of the transduction of the signals mediated by IGF-IRduring the attachment of IGFs.

FIGS. 3A, 3B and 3C: Recognition of native IGF-IR expressed on thesurface of MCF-7 cells by the monoclonal antibody 7C10.

For this experiment, the MCF-7 cells are incubated with the 7C10antibody or with a negative control antibody, then recovered with theaid of a fluorescent anti-species secondary antibody. The labeling isread on a FACS. The first histogram (FIG. 3A) corresponds to the MCF-7cells alone. In the second histogram (FIG. 3B), the unshaded curvecorresponds to the nonspecific labeling by a control isotype murineantibody. In the third histogram (FIG. 3C), the unshaded curve shows therecognition of IGF-IR by MAB 7C10.

FIGS. 4A, 4B and 4C: Labeling of Sf9 insect cells respectivelyexpressing IGF-IR or IR.

FIG. 4A shows the labeling of nontransfected cells alone (1) or cellslabeled with control commercial monoclonal antibodies respectivelyrecognizing IGF-IR (2) or IR (3). In FIG. 4B, Sf9 cells uniquelyexpressing IGF-IR are labeled with αIR3 (2) or anti-IR(3), the peak (1)representing the single cells. In FIG. 4C, Sf9 cells uniquely expressingIR are labeled with an anti-IR (3) or αIR3 (2), the peak (1)representing the single cells.

FIG. 5: Inhibitor effect of 7C10 antibody on the proliferation of MCF-7cells induced by IGF-I.

The MCF-7 cells are incubated in the presence of increasingconcentrations of IGF1 in the presence or in the absence of the MAB tobe tested. The cell proliferation is evaluated by following theincorporation of ³H thymidine. The commercial antibody αIR3 is used as apositive control of the experiment. The 7G3 is a murine anti-IGF-IR IgG1without activity on proliferation and used as a control isotype.

FIGS. 6A, 6B and 6C:

FIG. 6A: in vivo effect of the monoclonal antibody 7C10 on the growth ofMCF-7 tumors established in nude mice;

FIGS. 6B and 6C: Figures respectively from publications of Arteaga etal., (J. Clin. Invest., 84, 1418-1423, 1989) and from Li et al., (CancerImmunol. Immunother., 49, 243-252), and showing for FIG. 6B the effectof murine αIR3 (likewise written aIR3) and for FIG. 6C the effect of arecombinant scFv-Fc derived from the 1HH7 antibody on tumor growth.

FIG. 7: Comparative study of the effect of the MAb 7C10 and of tamoxifenon the growth in vivo of the tumor MCF-7.

FIGS. 8A, 8B, 8C and 8D: Study of the antitumor activity of the murineantibody 7C10 in different xenograft models of tumor cells in vivo.

FIG. 8A shows the results obtained on an osteosarcoma model SK-ES-1,FIG. 8B concerns an androgen-independent tumor of the prostate DU-145,FIG. 8C a model of non-small cell tumor of the lung A549 and FIG. 8D amodel of pancreatic cancer BxPC3.In these 4 models, the treatment wascarried out twice per week i.p. at a rate of 250 μg/dose/mouse. Thecurves 7G3, EC2 and 9G4 correspond respectively to three murine IgG1used as an experiment control isotype in each of the models.

FIG. 9: Study of the antitumor effect of the MAb 7C10 compared tonavelbine (vinorelbine) as well as the synergy of the two compounds onthe growth in vivo of the line A549.

FIG. 10: Comparative activity of MAb αIR3, 7C10 and 1H7 on the IGF2proliferation induced by MCF-7 cells.

FIG. 11: Comparison of the murine 7C10 and chimeric C7C10 MAb for theinhibition of the IGF1 proliferation of MCF-7 cells in vitro. Theantibody 9G4 is a murine IgG1 used as an experiment control isotype.

FIG. 12: Comparative effect of the 7C10 and h7C10 MAb (humanized 1,written here 7H2HM) on the in vitro model of IGF1-induced proliferationof MCF-7 cells.

FIG. 13: Effect of the 7C10 and h7C10 MAb (humanized 1, written here7H2HM) on the transduction of the signal induced by IGF1.The first lineof spots corresponds to the revelation, by an antiphospho-tyrosineantibody, of the phosphorylation of the immunoprecipitated β chain fromthe cells incubated in the presence of IGF1 alone or of IGF1 mixed withvarious antibodies to be tested. The 9G4 and the hIgG1 are respectivelythe control isotypes of the forms 7C10 and h7C10 (likewise written7H2HM). The second line of spots corresponds to the revelation of the βchain and shows that the quantity deposited in all of the wells isperfectly equivalent.

FIG. 14: Sequence of the cDNA (SEQ ID NO: 48), of its complementarystrand (SEQ ID NO: 50) and its translation into amino acids (SEQ ID NO:49), of the PCR fragment amplified from the mouse hybridoma 7C10 withthe primers MKV-1 (SEQ ID NO: 157) and MKC (SEQ ID NO: 158) and whichcodes for the 3′ end of the leader peptide and 7C10 VL. CDR regionsdisclosed as SEQ ID NOS: 1-6, respectively, in order of apppearance.

FIG. 15: Sequence of the cDNA (SEQ ID NO: 51), of its complementarystrand (SEQ ID NO: 53) and its translation into amino acids (SEQ ID NO:52), of the PCR fragment amplified from the mouse hybridoma 7C10 withthe primers MHV-12 (SEQ ID NO: 159) and MHC-1 (SEQ ID NO: 161), or MHV-8(SEQ ID NO: 160) and MHC-1 (SEQ ID NO: 161) and which codes for the 3′end of the leader peptide and 7C10 VH. CDR regions disclosed as SEQ IDNOS: 7-12, respectively, in order of apppearance.

FIG. 16: Recognition of the IGF-I receptor by the chimeric antibody7C10, likewise called C7C10 (supernatant of cos 7-transfected cellculture).

FIG. 17: Comparison of the amino acid sequence of mouse 7C10 VL (SEQ IDNO: 54) with cells of other mouse antibodies having the greatestsequence homology.

The numbering of the amino acids is that of Kabat et al., (1991). Theresidues in the framework regions (outside CDRs) which differ between7C10 VL and Kabat mouse subgroup II (SEQ ID NO: 57) are underlined. Adot indicates that the residue is identical at this position incomparison with the sequence of 7C10 VL. DRB1-4.3 (SEQ ID NO: 55)represents the sequence of the light chain of an anti-human mouseantibody MHC CLASS II B-Chain (access number in the Kabat databank isN011794). C94-5B11′CL (SEQ ID NO: 56) represents the sequence of thelight chain of a mouse antibody (access number in the Kabat databank isP019314).

FIG. 18: Comparison of amino acid sequences of mouse 7C10 VL (SEQ ID NO:54) with cells of human light chains belonging to Kabat human subgroupII (SEQ ID NO: 60) and having the greatest sequence homology.

The amino acid sequences are aligned and compared with that of mouse7C10 VL. A dot indicates that the residue is identical at this positionin comparison with the sequence of 7C10 VL. GM607 (SEQ ID NO: 58)represents the sequence of the kappa light chain secreted by the humanlymphoblastoid line GM607 (Klobeck et al., Nucleic Acids Res.,12:6995-7006, 1984a and Klobeck et al., Nature, 309:73-76, 1984b, theaccess number in the Kabat databank is N011606). DPK15/A19 (SEQ ID NO:59) represents the sequence of the human V germinal line kappa II.

FIG. 19: Comparison of amino acid sequences of variable regions of thelight chains (VL) of mouse 7C10 (SEQ ID NO: 54), of human antibody GM607 (SEQ ID NO: 58) and of two versions of humanized 7C10 1 and 2 (SEQID Nos. 61 and 65).

The amino acid sequences are aligned and compared with that of mouse7C10 VL. A dot indicates that the residue is identical at this positionin comparison with the sequence of 7C10 VL. GM607 represents thesequence of the kappa light chain secreted by the human lymphoblastoidline GM607 (Klobeck et al., 1984a and 1984b, access number in the Kabatdatabase: N011606).

FIG. 20: cDNA sequence (SEQ ID NO: 62), its complementary strand (SEQ IDNO: 64) and its translation into amino acids (SEQ ID NO: 63), of thegene constructed by de novo assembly coding for the leader peptide andthe humanized version 1 of 7C10 VL.

FIG. 21: cDNA sequence (SEQ ID NO: 66), its complementary strand (SEQ IDNO: 68) and its translation into amino acids (SEQ ID NO: 67), of thegene constructed by de novo assembly coding for the leader peptide andthe humanized version 2 of 7C10 VL.

FIG. 22: Comparison of the amino acid sequences of mouse 7C10 VH (SEQ IDNO: 69) with those of human mouse heavy chains belonging to Kabat mousesubgroup I(A) and having the greatest sequence homology.

The numbering of the amino acids is that of Kabat et al., (1991). Theresidues in the framework regions (outside CDRs) which differ between7C10 VH and Kabat mouse subgroup I(A) (SEQ ID NO: 71) are underlined. Adot indicates that the residue is identical at this position incomparison with the sequence of mouse 7C10 VH. AN03′CL (SEQ ID NO: 70)represents the sequence of the heavy chain of a mouse antibody (accessnumber in the Kabat databank: P001289).

FIG. 23: Comparison of amino acid sequences of mouse 7C10 VH (SEQ ID NO:69) with those of human heavy chains belonging to the Kabat humansubgroup II (SEQ ID NO: 72) and having the greatest sequence homology.

The underlined residues are part of the canonical structures defined byChothia et al., (1989). A dot indicates that the residue is identical atthis position in comparison with the mouse 7C10 VH sequence. Human VHFUR1′CL (SEQ ID NO: 73) represents the sequence of the heavy chain of ahuman anti-lamin B antibody IgM/K of autoimmune origin (Mariette et al.,Arthritis and Rheumatism, 36:1315-1324, 1993; access number in Kabat:N020619). Human germline (SEQ ID NO: 74) represents the sequence of thehuman germinal line 4.22 VH IV (Sanz et al., EMBO. J. 8:3741-3748,1989).

FIG. 24: Comparison of the amino acid sequences of the variable regionsof the heavy chains (VH) of mouse 7C10 (SEQ ID NO: 69) and of the threeversions humanized by CDR-grafting humanized VH 1, 2 and 3 (respectivelySEQ ID Nos. 75, 79 and 83). Human germline disclosed as SEQ ID NO: 162.

The numbering of the residues corresponds to that of Kabat. Thesequences are aligned and compared with that of mouse 7C10 VH. A dotindicates that the residue is identical at this position in comparisonwith the sequence of mouse 7C10 VH.

FIG. 25: cDNA sequence (SEQ ID NO: 76), its complementary strand (SEQ IDNO: 78) and its translation into amino acids (SEQ ID NO: 77), of thegene constructed by de novo assembly coding for the leader peptide andthe humanized version 1 of 7C10 VH.

FIG. 26: cDNA sequence (SEQ ID NO: 80), its complementary strand (SEQ IDNO: 82) and its translation into amino acids (SEQ ID NO: 81), of thegene constructed by de novo assembly coding for the leader peptide andthe humanized version 2 of 7C10 VH.

FIG. 27: cDNA sequence (SEQ ID NO: 84), its complementary strand (SEQ IDNO: 86) and its translation into amino acids (SEQ ID NO: 85), of thegene constructed by de novo assembly coding for the leader peptide andthe humanized version 3 of 7C10 VH.

FIG. 28: Comparison of the recognition activity of the IGF-I receptor bythe chimeric antibody 7C10 (called “C7C10”) and its humanized version 1(7C10 hum 1) in ELISA.

FIG. 29: Influence on the recognition activity of the IGF-I receptor ofthe humanized versions 1 and 2 of the light chain of the 7C10 antibodyin ELISA.

FIG. 30: Comparison of the recognition activity of the IGF-I receptor bythe chimeric antibody 7C10 and three humanized versions of the heavychain (7C10 hum 1, 2 and 3) in combination with humanized 7C10 VL 2 inELISA.

FIG. 31: Antitumor activity of the 7C10 antibody in an orthotopic modelA549.

FIGS. 32A, 32B, 32C and 32D: Study of the ADCC observed at the level ofA549 and MCF-7 cells cultured during 4 hours in the presence of theantibody 7H2HM (respectively FIGS. 32C and 32D). The antibody h4D5 isused in parallel as an experiment positive control for the cells A549and MCF-7 (respectively FIGS. 32A and 32B).

FIGS. 33A, 33B and 33C: Effects of the antibodies 7C10 and 7H2HM on thecell cycle of the MCF-7 cells.

FIG. 33A represents the proportion of MCF-7 cells in the G0/G1, S andG2/M phase in the absence of IGF1, expressed as a significant percentageof total MCF-7 cells observed.

FIG. 33B represents the proportion of MCF-7 cells in the G0/G1, S andG2/M phase in the presence of IGF1, expressed as a percentage of totalMCF-7 cells observed.

FIG. 33C represents the proportion of MCF-7 cells in the S (▪) and G2/M(□) phase, expressed as a percentage of total MCF-7 cells observed, inthe presence of the compounds indicated in the figure compared with acontrol sample in the absence of IGF1 (“0”).

FIGS. 34A and 34B: Comparative effect of the antibodies 7C10 and 7H2HMon the growth of A549 cells in vitro (FIG. 34A) and on the growth ofMCF-7 cells in vivo (FIG. 34B).

FIGS. 35A and 35B: Study of the synergy of the antibody 7H2HM combinedwith navelbine (NA) on the model A549 in vivo, compared with the controlsamples. FIG. 35A represents the development of the volume of theimplanted tumor as a function of the treatment carried out starting fromthe commencement of the treatment and over approximately 50 days (FIG.35A). FIG. 35B represents in a particular manner the results obtainedfor this development compared at approximately 48 days. In this figure,the results obtained with the antibody 7C10 have been introduced by wayof comparison (the asterisks (*) correspond to the comparison controlgroup/group (7C10+Na) or control group/group (7H2HM+Na) in a t-test).

FIG. 36: Study of the effect of the antibodies 7C10 and 7H2HM onapoptosis.

This figure represents the potentiation of the effect of doxorubicin bythe antibodies 7C10 and 7H2HM (doxorubicin 2 μg/ml).

FIGS. 37A to 37D: Demonstration by labeling in FACS of the presence ofEGFR and of IGF-IR on the surface of A549 cells.

FIG. 38: Effect of a coadministration of the MAB 7C10 and 225 on the invivo growth of the tumor A549.

FIG. 39: Effect of a coadministration of the MAB 7C10 and 225 on thesurvival of mice orthotopically implanted with A549 cells.

FIGS. 40A and 40B: Demonstration of the inhibition of tyrosinephosphorylation of the beta chain of IGF-IR and of IRS-1 by the MAB 7C10and 7H2HM.

FIG. 41: Demonstration of the induction of the internalization of IGF-IRby the MAB 7C10 and 7H2HM.

FIGS. 42A to 42C: Demonstration of the degradation of IGF-IR by the MAB7C10 and 7H2HM.

FIGS. 43A and 43B: Immuno-blotting with an anti-IGF-IR β-subunit andanti-IR β-subunit on filters containing cellular lysates obtained afterimmunoprecipitation and SDS-PAGE for two independent experiments (A andB).

FIG. 44: Immunocapture of R+ cell lysates IGF-IR in Maxisorb platescoated with 17-69 antibody and binding by ¹²⁵I-IGF-I in the absence orthe presence of increasing concentrations of unlabeled ligand (IGF-I) orantibodies (7C10, h7C10, 1H7, 9G4).

FIG. 45: Immunocapture of R-/IR-A cell lysates Hybrid-R^(A) in Maxisorbplates coated with 83-7 antibody and binding by ¹²⁵I-IGF-I in theabsence or the presence of increasing concentrations of unlabeled ligand(IGF-I) or antibodies (7C10, h7C10, 1H7, 9G4).

FIG. 46: Immunocapture of R-/IR-B cell lysates Hybrid-R^(B) in Maxisorbplates coated with 83-7 antibody and binding by ¹²⁵I-IGF-I in theabsence or the presence of increasing concentrations of unlabeled ligand(IGF-I) or antibodies (7C10, h7C10, 1H7, 9G4).

FIGS. 47A and 47B: Immuno-blotting analysis of antibody induceddegradation of the IGF-IR in A549 (A) and MCF-7 (B) cells.

FIG. 48: Immuno-blotting analysis of antibody degradation pathway ofIGF-IR in MCF-7 cells.

FIG. 49: Anti-tumoral activity of the murine antibody 7C10coadministrated with an anti-VEGF antibody on mice orthopicallyimplanted with A549 cells.

FIGS. 50 and 51: Comparison of the in vivo anti-tumoral activity of the7C10 and h7C10 antibodies on the A549 (FIG. 50) and MCF-7 (FIG. 51)models.

FIGS. 52 and 53: Comparison of the anti-leukemia activity of vinblastineand vincristine (FIG. 52) and of 4′R and 4′s deoxyvinblastines (FIG.53).

FIG. 54: In vivo antitumour activity of 4′R- and 4′S-deoxyvinblastinesconjugated with IGR-IR antibodies on human tumors of various origins.

FIG. 55: Schematic representation of the biosensor capturing assay. AMab directed against the constant Fc portion of either mouse or humanIgG1 were covalently attached onto a CM5 sensor surface. A limitedamount (400 RU) of Mab to be tested were immobilized and used to capturethe analyte hIGF-IR. The binding of Mab to the analyte is described bythe association and dissociation rate constants k_(a) and k_(d),respectively.

FIG. 56: Determination of h7C10 IC₅₀ in the IGF1 induced proliferativeassay. Results are expressed as proliferative indexes in panel A. PanelB shows an example of IC₅₀ calculation and panel C summarizes the IC₅₀data obtained with 5 different batches of h7C10 antibody.

FIG. 57: 7C10 causes rapid down-regulation of IGF-IR via the proteasomepathway (57 A). Lysosomal/endosomal pathways (57 B and C) seem also tobe involved in the antibody-induced down-regulation. Panel (57 D) showsthe low recovery of the IGF-IR after antibody treatment. In thesefigures 9G4 and 7G3 Mabs are respectively a irrelevant IgG1 antibody andan non neutralizing anti-IGF-IR antibody (IgG1 isotype).

FIG. 58: staining of either tumoral or normal tissues from lung andbreast cancer patients. Comparison with tissues from normal regions.

FIG. 59: h7C10 down regulation of IGF-IR in vivo. Nine Swiss Nude micebearing a MCF-7 xenograft tumor were studied. Tumors from 3 mice wereremoved before treatment (lanes 1-3), 3 mice were treated with h7C10(lanes 4-6) and 3 mice received a human isotype control (IgG1). Lane 9is not interpretable because of the low amount of protein loaded (CK19).

FIG. 60: Determination of h7C10 IC₅₀ in the IGF2 induced proliferativeassay. Results are expressed as proliferative indexes in panel A. PanelB shows an example of IC₅₀ calculation and panel C summarizes the IC₅₀data obtained with 5 different batches of h7C10 antibody.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat same are intended only as illustrative and in nowise limitative. Insaid examples to follow, all parts and percentages are given by weight,unless otherwise indicated.

EXAMPLE 1 Generation and Selection of the Murine Monoclonal Antibody(MAb)

With the aim of generating MAb specifically directed against IGF-IR andnot recognizing the IR, a protocol comprising 6 screening stages wasenvisaged.

It consisted in:

immunizing mice with recombinant IGF-IR, in order to generatehybridomas,

screening the culture supernatants by ELISA on the recombinant proteinwhich served for immunization,

testing all the supernatants of hybridomas positive by ELISA on thenative receptor overexpressed on the surface of MCF-7 tumor cells,

evaluating the supernatants of hybridomas positive in the two firstscreenings in terms of differential recognition of IGF-IR and of IR oninsect cells infected with baculoviruses respectively expressing IGF-IRor IR,

verifying that the antibodies selected at this stage were capable ofinhibiting in vitro the induced IGF1 proliferation of the MCF-7 cells,

ensuring the in vivo activity, in nude mice, of the candidate retainedin terms of impact on the growth of the tumor MCF-7.

All of these different stages and results obtained will be brieflydescribed below in example 1.

For the immunization stage, mice were injected twice, by thesubcutaneous route, with 8 μg of recombinant IGF-IR. Three days beforethe fusion of the cells of the female rat with the cells of the murinemyeloma Sp2OAg14, the mice were stimulated by an intravenous injectionof 3 μg of the recombinant receptor. Fourteen days after the fusion, thesupernatants of hybridomas were screened by ELISA, on plates sensitizedby recombinant IGF-IR. The hybridomas whose supernatants were foundpositive were conserved and amplified before being tested on the FACScanso as to verify that the antibodies produced were likewise capable ofrecognizing native IGF-IR. In order to do this, MCF-7 cells from anestrogen-dependent tumor of the breast overexpressing IGF-IR wereincubated with each of the culture supernatants produced by thehybridomas selected in ELISA. The native/MAb receptor complexes on thesurface of the cell were revealed by a secondary anti-species antibodycoupled to a fluorochrome. FIGS. 3A to 3C show a histogram type obtainedwith the supernatant of the hybridoma 7C10 (FIG. 3C) compared with acell labeling alone+secondary antibody (FIG. 3A) or with a labelingutilizing a control isotype (FIG. 3B).

At this stage of the selection, only the hybridomas secreting MAb at thesame time recognizing the recombinant receptor and the native receptorwere selected and cloned. The MAb secreted by these hybridomas wereproduced and then purified before being tested on the FACScan, accordingto the method described above, on Sf9 insect cells expressing IGF-IR orIR in order to eliminate the hybridomas at the same time recognizing thetwo receptors. FIG. 4A shows a total recovery of the histograms 1, 2, 3respectively corresponding to the noninfected cells+secondary antibodies(1), to the noninfected cells labeled by αIR3+secondary antibodies (2)and to the noninfected cells labeled by an anti-IR antibody+secondaryantibodies (3). This first result shows well the absence of IGF-IR andof IR detectable on the surface of these noninfected insect cells. FIG.4B shows a labeling of infected cells by a baculovirus expressingIGF-IR. In this second figure, the αIR3, used as a positive control,labels well, as expected, the cells (peak 2), while the anti-IR (peak 3)is superimposed on the peak of single cells. Finally, in FIG. 4C, it isshown that the anti-IR labels well, as expected, the Sf9 cellsexpressing the IR (peak 3), but in an unexpected manner, the αIR3described in the literature as specific for IGF-IR seems likewise torecognize the IR (peak 2).

The results obtained in this third screening system are summarized intable 1 and show the generation of an MAb: 7C10, satisfying the criteriaof recognition of the IGF-IR and of nonrecognition of the IR. Theisotyping of the Mab 7C10 has shown that it involves an IgG1.

TABLE 1 Comparative reactivity of MAb 7C10 on Sf9 insect cellsexpressing IGF-IR or IR: MFI (Mean fluorescence intensity) Noninfectedcells IGF1R + cells IR + cells Cells 8 8 7 Anti-IR 4.6 9 91 Anti-IGF-IR(αIR3) 9 35 32 EC2 8 13 11 Anti-mouse FITC 4.3 9 13 UltraCulture medium9 10 11 15B9 7.5 25 77.8 9F5D 8 41 40 13G5 7.8 37 24 7C10 8.6 49 13

The two last screenings provided for the selection of the MAb consistedin verifying that the latter was very capable of inhibiting the cellproliferation induced by the IGF-I in vitro and in vivo on the cell lineMCF-7.

For the in vitro selection, the MCF-7 cells were inoculated, deprived offetal calf serum, then incubated in the presence of increasingconcentrations of IGF-I (from 1 to 50 ng/ml) in the presence or in theabsence of the 7C10 antibody to be tested added to a final concentrationof 10 μg/ml. In this experiment, the commercial αIR3 MAb was introducedas a positive control and the 7G3 MAb (isolated in parallel to the 7C10and weakly recognizing the native receptor (MFI on the FACS of 50compared with 200 for the MAb 7C10)) as a control isotype. The cellproliferation is estimated by following on the β counter theincorporation of tritiated thymidine by the cells. The results areexpressed as a proliferative index. The data presented in FIG. 5 showthat IGF1 is capable of stimulating in a dose-dependent manner theproliferation of the MCF-7 cells. The MAb αIR3, used as a positivecontrol, completely inhibits the proliferation of the MCF-7 cellsinduced by the IGF-I. In the same manner, the MAb 7C10 significantlyinhibits the growth of the MCF-7 cells induced by IGF-I. Finally, theMAb 7G3 used as an isotype control turns out well, as expected, withouteffect on the tumor cell growth in vitro of the MCF-7 cell.

The in vivo selection was carried out in an established tumor model. Inorder to do this, nude mice received a subcutaneous implant ofslow-release estrogen, indispensable for the taking of the tumor in amurine model. Twenty-four hours after implantation of the estrogens,5.10⁶ MCF-7 cells are grafted onto the right flank of the mousesubcutaneously. Five days after this cell graft, the tumors aremeasurable and batches of 6 mice are formed at random. The treatment ofthe mice is carried out twice per week, during 5 to 6 weeks, at the doseof 250 μg/dose/mouse. In the control group, the mice are treated in thesame fashion with a murine control isotype. The results presented inFIG. 6A show a very significant inhibition of the tumor growth inducedby the antibody 7C10.This activity is particularly unexpected ifreference is made to the data available concerning αIR3, always used asa reference in the domain of the receptor for IGF1, and known for nothaving any activity in vivo on the growth of estrogen-dependent tumors(see FIG. 6B). In the same way, compared with the results obtained withthe recombinant antibody scFv-Fc derived from the murine MAb 1H7 (seeFIG. 6C), the MAb 7C10 is much more efficacious in the in vivoinhibition of the growth of the MCF-7 cells. This difference of activitycould be realty, without any limitation, to some particular propertiesof 7C10 and h7C10 antibodies which recognize the hybrid-R, isoform(s) Aand/or B, (see example 26). Another non exclusive hypothesis could bethe recognition by these antibodies of the atypical IGF-IR as describedin the paper of Siddle et al., (Siddle et al., 1994, Horn Res. 41(suppl.2): 56-65).

Equilibrium dissociation constants (K_(D)) of a series of 7C10 and h7C10antibodies directed against the extracellular domain of humaninsulin-like growth factor-1 receptor (hIGF-IR) were calculated by theratio between dissociation and association rate constants, as defined bysurface plasmon resonance using a BIAcore X instrument. Capture of theinvestigated antibodies was used to favor a proper presentation of theirantigen-binding site (FIG. 55). Therefore, a mixture of goat anti-humanIgG Fc and rabbit anti-mouse IgG Fc polyclonal antibodies werecovalently linked on both flowcells (FC1 and FC2) of a CM5 sensorchip.Typically 300 to 400 resonance units (RU) of each anti-hIGF1-R Mab werecaptured on FC2 (FC1 being a reference cell to evaluate non specificinteractions of the analyte with the matrix). The analyte, correspondingto the extracellular domain of hIGF1-R, was tested at 5 differentconcentrations ranging from 12.5 to 200 nM at 25° C. at a flow rate of30 μl/min. Under this experimental set-up, a mean K_(D) of 0.86±0.09 nMwas obtained for 3 different batches of A2CHM Mab. These values wereclosed to that obtained for the mouse Mab 7C10 (K_(D): 0.86±0.14 nM)Table 2.

TABLE 2 Kinetic rate constants and equilibrium dissociation constant forthe interaction of hIGF-IR with a series of immobilized monoclonalantibodies: k_(a) × Antibodies 10⁻⁵ M⁻¹s⁻¹ k_(d) × 10⁴ s⁻¹ K_(D) × 10⁹ MX² 7C10 1.66 ± 0.01 1.49 ± 0.03 0.897 ± 0.022 0.631 batch 7 1.82 ± 0.011.39 ± 0.03 0.765 ± 0.023 1.00 1.66 ± 0.01 1.52 ± 0.03 0.920 ± 0.0220.764 Mean  1.71 ± 0.16*  1.47 ± 0.11*  0.86 ± 0.14** h7C10 1.11 ± 0.010.984 ± 0.030 0.886 ± 0.036 0.409 Batch F50035 1.13 ± 0.01 0.953 ± 0.0290.844 ± 0.034 0.407 BR2.113B 1.12 ± 0.01 0.974 ± 0.029 0.872 ± 0.0340.396 Mean  1.12 ± 0.02*  0.97 ± 0.03*  0.87 ± 0.04** h7C10 1.04 ± 0.010.767 ± 0.030 0.736 ± 0.039 0.344 Batch F50035 1.00 ± 0.01 0.827 ± 0.0300.827 ± 0.038 0.381 BR2.121A 1.01 ± 0.01 0.837 ± 0.030 0.826 ± 0.0380.395 Mean  1.02 ± 0.04*  0.81 ± 0.06*  0.80 ± 0.09** h7C10 1.12 ± 0.010.967 ± 0.030 0.863 ± 0.034 0.403 Batch F50035 1.14 ± 0.01 1.04 ± 0.030.913 ± 0.035 0.464 BR2.113A 1.12 ± 0.01 1.04 ± 0.03 0.927 ± 0.035 0.470Mean  1.13 ± 0.02*  1.02 ± 0.07*  0.90 ± 0.08** Rate constants k_(a) andk_(d) were determined for each data set by using the global fittingalgorithm described in the BIA evaluation 3.1 software and based on a1:1 Langmuir binding model without incorporating a mass transport term.Quality of the global fitting to the experimental data is indicated bythe χ² value. *The 95% confidence intervals of the kinetic rates werecalculated using the formula SE = σ/3^(1/2) × 2.920. **The confidenceintervals of the K_(D) were calculated using the formula SE(K_(D)) =[SE(k_(a)) × k_(d) + k_(a) × SE(k_(d))]/k_(a) ².

EXAMPLE 2 Comparison of the Effect of 7C10 and of Tamoxifen on the invivo Growth of the Tumor MCF-7

With the aim of determining the effectiveness of the treatment by theantibody 7C10 in the context of estrogen-dependent cancer of the breast,7C10 was compared with the tamoxifen compound currently used for thetreatment of mammary carcinoma in the context of developed forms withlocal and/or metastatic progression and in the context of the preventionof recurrences (see VIDAL 2000, pages 1975-1976).

In hormone-dependent cancers of the breast, a significant correlationexists between the expression of the receptors for estrogens (ER) andthat of the IGF-IR (Surmacz E. et al., Breast Cancer Res. Treat.,February, 47(3):255-267, 1998). Furthermore, it seems that the estrogens(E2) act in synergy with IGF1 (sometimes written IGF-I or IGFI) in orderto stimulate cell proliferation. It has in effect been shown that atreatment with E2 increases by approximately 10 times the mRNA level ofIGF-IR as well as the expression level of the protein (Lee A. V. et al.,Mol. Endocrinol., May, 13(5):787-796, 1999). This increase is manifestedby a significant increase in the phosphorylation of the IGF-IR. Inaddition, the E2 significantly stimulates the expression of IRS-1(“IRS-1” for “Insulin Receptor Substrate-1”) which is one of thesubstrates of the phosphorylated IGF-IR.

Tamoxifen has been widely used for many years in hormone therapy for thetreatment of patients suffering from E2-dependent breast cancers (ForbesJ. F., Semin. Oncol., Feb., 24 (1st Suppl. 1):S1-5-S1-19, 1997). Thismolecule enters into competition with the estradiol and inhibits theattachment of this to its receptor (Jordan V. C., Breast Cancer Res.Treat., 31(1):41-52, 1994). It has in addition been demonstrated thattamoxifen is capable of inhibiting the IGF-IR-dependent proliferation byinhibiting the expression of the receptor and its phosphorylation(Guvakova M. A. et al., Cancer Res., Jul. 1, 57(13):2606-2610, 1997).These data as a whole seem to indicate that IGF-IR is an importantmediator of the proliferation induced by the E2/ER interaction.

The long-term use of tamoxifen is associated with a significant increasein the risk of endometrial cancer (Fisher et al., J. of National CancerInstitute, 86, 7:527-537, 1994; VIDAL 2000, 1975-1976) and of collateralrecurrence of E2-independent cancer of the breast (Li C. I. et al., J.Natl. Cancer Inst., Jul. 4, 93(13):1008-1013, 2001). In this context, acomparison of the in vivo antitumor effect of the antibody 7C10 and oftamoxifen has been carried out on the MCF-7 model so as to determine thepart of the activity connected with IGF-IR in the mediated ERproliferation. In order to do this, 7.10⁶ MCF-7 cells were implanted sc(subcutaneously) in nude mice, 24 hours after implantation in these samemice of a grain of estradiol with prolonged release (0.72 mg/tabletliberated over 60 days), indispensable for the establishment of anyE2-dependent human tumor in this animal species. Five days after thisimplantation, the tumors are measured and groups of 6 mice are formed.These groups are treated respectively with 1) the 7C10 antibody injectedip (intraperitoneally) at a rate of 250 μg/mouse, twice per week, 2) 10μg of tamoxifen taken in PBS containing 3% of hydroxypropyl-cellulose(HPC) ip or 3) the solvent in which the tamoxifen is taken up(hydroxypropylcellulose). The tamoxifen is administered daily for 4weeks except at the weekend. The mice treated with the MAb 7C10 likewisedaily receive an injection of PBS with 3% HPC. A study was previouslycarried out in order to verify that the solvent alone is withoutinfluence on the tumor growth.

The results presented in FIG. 7 shown that the MAb 7C10 is capable ofsignificantly inhibiting the growth of the tumor MCF-7 in vivo (theasterisks (*) correspond to the comparison control group/7C10 group in at-test). In a surprising fashion, the antibody 7C10 seems to besignificantly more efficacious than tamoxifen for the inhibition of thetumor growth (the circles (°) correspond to the comparison tamoxifengroup/7C10 group in a t-test) suggesting that this type of treatment byMAB might be substituted for treatment with tamoxifen.

EXAMPLE 3 Demonstration of the Antitumor Activity of the MAb 7C10 invivo on Human Tumors of Different Origins

a) In vivo Activity of the Antibody 7C10 in 4 Tumor Models:

In order to generalize the activity of the 7C10 antibody to other tumorsexpressing the receptor for IGF1, 7C10 was tested in vivo in anandrogen-independent model of tumor of the prostate DU145 (likewisewritten DU-145), in an SKES-1 osteosarcoma model, in a model ofnon-small cell tumor of the lung A549 and in a model of pancreaticcancer BxPC3.The protocol is comparable to that described above forMCF-7 and the results presented in FIGS. 8A to 8D show a significantactivity of this MAB in the 4 tumor models. The activity observed in themodel of tumor of the prostate is to be noted very particularly inasmuchas the single chain scFv of the MAB 1H7 is without activity in anandrogen-independent model of tumor of the prostate (Li et al., 2000).

b) In vivo Activity of the Antibody 7C10 in an Orthotopic Model A549:

The conventional xenograft models as described above do not allow thestudy of drugs on metastatic dissemination. In effect, the tumorsimplanted s.c. (subcutaneously) remain localized at the sight ofinjection and are therefore not really a reflection of the situation inman. In order to evaluate our antibody in a model closer to reality, theA549 cells were implanted in an intrapleural location. This model, whichis well described (Clin. Cancer Res. 2000 Jan.; 6(1):297-304) allows ametastatic dissemination close to that observed in man to be observed,with mediastinal, pulmonary, cardiac and vertebral metas-tases. In thestudy which was carried out, 10⁶ A549 cells were injected intrapleurallyinto female nude mice. 7 days after implantation, the mice were dividedinto 2 batches of 22.One of these batches received a challenge dose of500 μg/mouse and was then treated twice per week at a rate of 250 μg of7C10/dose. The second batch was treated according to the same schemewith the control isotype 9G4.FIG. 31 shows a significant extension ofsurvival in the mice treated with the MAB 7C10 indicating that thisantibody is capable of having an action on metastatic dissemination.

EXAMPLE 4 Comparison of the MAb 7C10 with Navelbine in vivo; Effect of aCoadministration of the two Treatments

Navelbine is a chemotherapy compound indicated in non-small cell cancerof the lung and in metastatic cancer of the breast. The comparativestudy of 7C10 and of navelbine and the possible synergy between the twoproducts was studied on the tumor model A549.For this study, 5.10⁶ A549cells were grafted subcutaneously on the right flank of the mouse. Fivedays after the cell graft, the tumors are measurable and the treatmentswith MAb and/or navelbine are commenced. The MAb dose is always 250μg/dose/mouse, twice per week, intra-peritoneally. Concerning navelbine,it will be administered at the maximum dose tolerated by the mouse or 10mg/kg, intraperitoneally. For this treatment three injections will becarried out at intervals of 7 days. During the coadministrations, thetwo products are mixed before injection.

The results presented in FIG. 9 show in a surprising fashion that, inthis model, the antibody 7C10 is as active as the conventional treatmentwith navelbine. A very significant synergy of the two products islikewise observed with five mice out of seven not having measurabletumors on day 72.

EXAMPLE 5 Study of the in vitro Inhibition of the IGF2-Induced Growth ofthe MCF-7 Tumors

As indicated above, IGF-IR is overexpressed by numerous tumors but ithas furthermore been described that in a good part of the cancers of thebreast and of the colon especially, the proliferation signal is given tothis receptor via IGF2 (sometimes written IGF-II or IGFII). It istherefore essential to ensure that the MAb 7C10 is likewise capable ofinhibiting the IGF2 growth induced on the MCF-7 tumor in vitro. In orderto do this, cells were inoculated into 96-well plates, deprived of fetalcalf serum and stimulated by the addition of 200 ng of IGF2 per ml,final concentration, of medium, in the presence and in the absence ofthe MAb to be tested introduced at a concentration of 10 μg/ml. Theresults presented in FIG. 10 show that IGF2, like IGF1, significantlystimulates the growth of MCF-7 cells. The addition of a control isotype,9G4, remains without effect on this stimulation. As already described byDe Léon et al., (Growth Factors, 6:327-334, 1992), no effect is observedduring the addition of the MAb αIR3.On the other hand, 7C10 totallyinhibits the growth induced by IGF2.Its activity is significantly betterthan that of 1H7.

EXAMPLE 6 Biological Activity of the Chimeric 7C10 (C7C10) and Humanized(h7C10) Antibodies 7C10

a) 7C10/C7C10 and 7C10/h7C10 Comparison on the MCF-7 Model in vitro:

The chimeric form of the MAb 7C10 and the purified humanized form 1(written here 7H2HM) were tested in vitro in the MCF-7 model asdescribed above. The results presented respectively in FIGS. 11 and 12show that these two forms have perfectly preserved their properties ofinhibiting the IGF1-induced growth of the MCF-7 tumor.

To determine the IC₅₀ values of the h7C10 antibody a dose range ofantibodies from various batches has been tested in vitro in the IGF1induced proliferative assay. The results described in FIG. 56demonstrate that all the tested batches are comparable and display andIC₅₀ value close to 0.3 nM. These data are in agreement with the K_(D)values determined by Biacore analysis and described at the end ofexample 1.

To determine the IC₅₀ values of the h7C10 antibody a dose range ofantibodies from various batches has been tested in vitro in the IGF2induced proliferative assay. The results described in FIG. 60demonstrate that all the tested batches are comparable and display andIC₅₀ value close to 0.3 nM. These IC₅₀ values are close to the oneobtained in the IGF1 induced proliferative assay described above.

b) Comparative Effect of the MAb 7C10 and h7C10 on the Transduction ofthe Signal Induced by the Attachment of IGF1 to its Receptor:

The activity of the inhibition of the IGF1 growth induced in vitro onthe line MCF-7 ought to be the translation of an inhibition of thetransduction of the signal mediated by IGF1 during the attachment of theMAb 7C10 to the receptor. In order to verify this hypothesis, MCF-7cells were incubated with or without IGF1, in the presence or in theabsence of the antibodies to be tested. After a short incubation time,the cells were lyzed, the β chain immunoprecipitated and thephosphorylation of this subunit estimated with the aid of anantiphosphotyrosine kinase antibody. The results presented in FIG. 13show that the attachment of the 7C10 or of the h7C10 significantlyinhibits the phosphorylation of the β subunit of IGF-IR contrary to anirrelevant murine (9G4) or human antibody (written IgG1 on the scheme).

c) Involvement of the 7H2HM Antibody in the Mechanisms of ADCC:

The inhibition of the transduction of the signal described above inparagraph b) is the principal mechanism of action involved in thebiological activity of the antibodies 7C10 and 7H2HM. It is, however,probable that during its administration in man, the antibody 7H2HM, ofisotype IgG1, is capable of inducing cell lysis by a mechanism of ADCCtype (Antibody Dependent Cellular Cytotoxicity). In order to verify thispoint, NK (Natural Killer) cells coming from the peripheral blood ofhuman donors are placed in the presence of A549 or MCF-7 cellspreviously incubated for 4 hours with 10 μg of 7H2HM antibody per 5.10⁵cells and labeled with ⁵¹Cr (50 μg). In this experiment, herceptin(written h4D5 on FIGS. 32A and 32B) is used as an experiment positivecontrol. FIGS. 32A to 32D show that, as expected, herceptin induces asignificant ADCC on the two cells A549 and MCF-7 (see respectively FIGS.32A and 32B). 7H2HM is likewise capable of inducing an ADCC on the A549cells (see FIG. 32C), but this phenomenon is of smaller amplitude on theMCF-7 cells (see FIG. 32D).

Regarding the present example, it would be evident for a man skilled inthe art to test, in the same way, other effector functions such as, forexample, CDC.

d) Effects of the Antibodies 7C10 and 7H2HM on the Cell Cycle:

The inhibition of the cell growth observed in vitro on the line MCF-7should be manifested by an effect on the cell cycle. In order to replyto this question, 4.10⁵ cells are inoculated into 6-well plates. 24hours after inoculation, the calf serum is removed and IGF1 added in thepresence or in the absence of the antibodies to be tested. Afterincubation for 24 hours, the cells are recovered for the study of thecell cycle. FIG. 33B demonstrates the effect of IGF1 on the entry intothe cycle and the growth of the MCF-7 cells compared with the entry intothe cycle and the growth of the MCF-7 cells in the absence of IGF1 (seeFIG. 33A). After addition of the growth factor, a significant decreasein the G0/G1 phase (from 88.2% to 56.3%) to the benefit of the S (from7.8% to 31%) and G2/M (from 4% to 12.7%) phases is observed. During theaddition of the antibodies 7C10 and 7H2HM (see FIG. 33C), a significantinhibition of the entry into the cycle is observed. In it is to be notedthat the murine antibody and its humanized homolog have a comparableactivity on the cell cycle. The αIR3, introduced as a positive control,seems slightly less active than the 7C10 and the 7H2HM in this test. Theantibody 9G4 used as a control isotype is without effect on the cellcycle.

e) Comparative Activity in vivo of the Antibodies 7C10 and 7H2HM on theModel A549:

In order to confirm the activity of the humanized antibody 7H2HM invivo, the latter was compared with 7C10 in the model of non-small celltumor of the lung A549.This experiment was carried out exactly asdescribed above except for the dose of antibody which is 125 μg/dosetwice per week in place of 250 μg/dose twice per week and that of thefact of the nonavailability of great quantities of 7H2HM. The antibody9G4 was used as an isotype control for 7C10 and an irrelevant humanimmunoglobulin of isotype IgG1 (below called HIgG1) was used as acontrol for the humanized antibody 7H2HM.

FIG. 34A shows that there are no significant differences between the 9G4and HIgG1 control curves. As expected, a significant inhibition of thetumor growth is observed with the murine antibody 7C10.Concerning thehumanized antibody 7H2HM, the activity observed is of exactly the sameintensity as that observed with its murine counterpart. This data, inaddition to the observations described above in vitro, indicates thatthe humanization has not modified the properties of the antibodygenerated. On the other hand, in the xenograft models in the mouse, theactivity of the humanized antibody seems to be integrally connected witha mechanism of inhibition of the transduction of the signal. In effect,if an ADCC part was in play in the inhibition of the tumor growth in theNude mouse, a difference ought to be observed between the activity ofthe murine and humanized antibodies.

An in vivo experiment was likewise carried out on the MCF-7 breast tumormodel and shows that, as expected, the antibody 7H2HM is perfectlycomparable with the murine antibody 7C10 for the inhibition of thegrowth of this tumor in vivo (FIG. 34B).

f) Demonstration of a Synergy Between the 7H2HM and Navelbine:

The protocol described in example 4 was repeated with the aim ofreproducing the results obtained with 7C10 with its humanized homolog:the antibody 7H2HM.

The results presented in FIGS. 35A and 35B show that, as in the case of7C10, a significant synergy is demonstrated between the humanizedantibody 7H2HM and navelbine.

g) Effect of the Antibodies 7C10 and 7H2HM on the Apoptosis of MCF-7Cells in vitro:

As indicated above, IGF-IR is capable of conferring protection againstapoptosis when it is overexpressed on the surface of cells. Furthermore,it has been demonstrated in these examples that the antibodies 7C10 and7H2HM were capable of potentiating an active compound in chemotherapy.In order to test the power of the antibodies 7C10 and 7H2HM to induceapoptosis, and to explain in part their synergy potential with thechemotherapy, experiments were conducted on the MCF-7 cells in thepresence or in the absence of doxorubicin, a medicament known to inducethe apoptosis of this cell line in vitro. In these experiments, theMCF-7 cells are inoculated at 2.10⁴/cm² in Petri dishes and cultured for24 h in RPMI without phenol red supplemented with 10% of fetal calfserum (FCS). The cells are then washed twice with PBS and put back intoculture in medium with 0% FCS. They are allowed an adaptation time of 10minutes at 37° C. before the addition of the antibodies at 10 μg/ml.After an extra 10 minutes at 37° C., recombinant IGF-I (Sigma) is addedto the culture medium to a final concentration of 50 ng/ml. The cellsare left at 37° C. again for one hour in order to allow the attachmentof the antibodies and of the IGF-I. Finally, the doxorubicin (Sigma) isadded to the culture medium at 2 μg/ml and the cells are incubated for24 hours at 37° C.

The experiments have likewise been conducted with navelbine at aconcentration of 10 μg/ml.

The analysis of the cell viability is carried out by flow cytometricanalysis after labeling with the annexin V-FITC (20 minutes, 4° C.) andDAPI (2 μg/ml). The percentage of dead cells considered is the labeledpopulation Annexin+/DAPI+. The antibody 5C2 is used as a controlisotype.

The results represented in FIG. 36 show that doxorubicin inducesapoptosis in 8% of the MCF-7 cells. When the cells are treatedconjointly with the antibody 7C10 and the doxorubicin a significantincrease in cell death is observed. The same effect is shown with theantibody 7H2HM. The same type of results was observed when the antibodyis combined with navelbine.

EXAMPLE 7 Cloning Strategy of Genes Coding for the Variable Regions ofthe Heavy and Light Chains of the Monoclonal Antibody (MAb) 7C10

The total RNA was extracted from 10⁷ cells of hybridomas secreting theantibody 7C10 by using the TRI REAGENT™ (according to the instructionsgiven by the supplier, SIGMA, T9424). The first cDNA strand wassynthesized with the aid of the ‘First strand cDNA synthesis’ kit ofAmersham-Pharmacia (#27-9621-01, according to the instructions given bythe supplier). For the two chains, the reaction was primed with theoligonucleotide Not I-d(T)18, comprised in the Kit.

The cDNA:mRNA hybrid thus obtained was used for the amplification by PCRof the genes coding for the heavy and light chains of the Mab 7C10.ThePCR were carried out by using a combination of oligonucleotides specificfor the heavy and light (Kappa) chains of mouse immunoglobulins. Theprimers corresponding to the 5′ ends hybridize in the regioncorresponding to the signal peptides (Table 3 for heavy chains, Table 4for light chains). These primers were compiled from a large number ofmouse antibody sequences found in the databanks (Jones S. T. et al.,Bio/Technology 9:88-89, 1991). The primers corresponding to the 3′ endshybridize in the constant regions of the heavy chains (CH1 domain of thesubclass IgG1, not far from the V-C junction, MHC-1 primer Table 5) andlight chains (Kappa domain not far from the V-C junction, MKC primerTable 5).

TABLE 3 Oligonucleotide primers for the 5′ regionof the variable domains of the heavy chainsof mouse immunoglobulin (MHV) (“MHV” for “Mouse Heavy Variable”): MHV-1:5′ ATGAAATGCAGCTGGGTCATSTTCTT 3′ (SEQ ID NO: 13) MHV-2: 5′ATGGGATGGAGCTRTATCATSYTCTT 3′ (SEQ ID NO: 14) MHV-3: 5′ATGAAGWTGTGGTTAAACTGGGTTTT 3′ (SEQ ID NO: 15) MHV-4: 5′ATGRACTTTGGGYTCAGCTTGRT 3′ (SEQ ID NO: 16) MHV-5: 5′ATGGACTCCAGGCTCAATTTAGTTTT 3′ (SEQ ID NO: 17) MHV-6: 5′ATGGCTGTCYTRGSGCTRCTCTTCTG 3′ (SEQ ID NO: 18) MHV-7: 5′ATGGRATGGAGCKGGRTCTTTMTCTT 3′ (SEQ ID NO: 19) MHV-8: 5′ATGAGAGTGCTGATTCTTTTGTG 3′ (SEQ ID NO: 20) MHV-9: 5′ATGGMTTGGGTGTGGAMCTTGCTATT 3′ (SEQ ID NO: 21) MHV-10: 5′ATGGGCAGACTTACATTCTCATTCCT 3′ (SEQ ID NO: 22) MHV-11: 5′ATGGATTTTGGGCTGATTTTTTTTATTG 3′ (SEQ ID NO: 23) MHV-12: 5′ATGATGGTGTTAAGTCTTCTGTACCT 3′ (SEQ ID NO: 24) NB KEY: R = A/G, Y = T/C,W = A/T, K = T/G, M = A/C, S = C/G.

TABLE 4 Oligonucleotide primers for the 5′ regionof the variable domains of kappa (light) chainsof mouse immunoglobulin (MKV) (“MKV” for “Mouse Kappa Variable”): MKV-1:5′ ATGAAGTTGCCTGTTAGGCTGTTGGTGCT 3′ (SEQ ID NO: 25) MKV-2: 5′ATGGAGWCAGACACACTCCTGYTATGGGT 3′ (SEQ ID NO: 26) MKV-3: 5′ATGAGTGTGCTCACTCAGGTCCT 3′ (SEQ ID NO: 27) MKV-4: 5′ATGAGGRCCCCTGCTCAGWTTYTTGG 3′ (SEQ ID NO: 28) MKV-5: 5′ATGGATTTWCAGGTGCAGATTWTCAGCTT 3′ (SEQ ID NO: 29) MKV-5A: 5′ATGGATTTWCARGTGCAGATTWTCAGCTT 3′ (SEQ ID NO: 30) MKV-6: 5′ATGAGGTKCYYTGYTSAGYTYCTGRG 3′ (SEQ ID NO: 31) MKV-7: 5′ATGGGCWTCAAGATGGAGTCACA 3′ (SEQ ID NO: 32) MKV-8: 5′ATGTGGGGAYCTKTTTYCMMTTTTTCAAT 3′ (SEQ ID NO: 33) MKV-9: 5′ATGGTRTCCWCASCTCAGTTCCTT 3′ (SEQ ID NO: 34) MKV-10: 5′ATGTATATATGTTTGTTGTCTATTTC 3′ (SEQ ID NO: 35) MKV-11: 5′ATGGAAGCCCCAGCTCAGCTTCTCTT 3′ (SEQ ID NO: 36) MKV-12A:  5′ATGRAGTYWCAGACCCAGGTCTTYRT 3′ (SEQ ID NO: 37) MKV-12B:  5′ATGGAGACACATTCTCAGGTCTTTGT 3′ (SEQ ID NO: 38) MKV-13: 5′ATGGATTCACAGGCCCAGGTTCTTAT 3′ (SEQ ID NO: 39) NB KEY: R = A/G, Y = T/C,W = A/T ,K = T/G, M = A/C, S = C/G.

TABLE 5 Oligonucleotide primers for the 3′ ends of themouse V_(H) and V_(L) genes: Light chain (MKC): 5′ACTGGATGGTGGGAAGATGG 3′ (SEQ ID NO: 40)Constant region of the mouse Kappa domain:A D  A  A P  T  V S I F  P  P S  S (SEQ ID NO: 41)GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA CCA TCC (SEQ ID NO: 42) AGT   || ||| ||| ||| ||| ||| ||| (MKC) CC ATC TTC CCA CCA TCC AGT(SEQ ID NO: 43) Heavy chain (MHC-1) 5′ CCAGTGGATAGACAGATG 3′(SEQ ID NO: 44) CH1 domain of mouse gamma-1 (IgG1 subclass):A K  T  T P  P S  V Y P  L (SEQ ID NO: 46)GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG (SEQ ID NO: 45)     ||| |||||| ||| ||| ||| ||| (MHC-1) CCC CCA TCT GTC TAT CCA CTG (SEQ ID NO: 47)

EXAMPLE 8 Sequences of Immunoglobulins Cloned from the Mouse Hybridoma7C10

By following the amplification strategy described above, PCR productscorresponding to the variable regions of the heavy (VH) and light (VL)chains were cloned by using the “pGEM®-T Easy Vector Systems” (Promega).For 7C10 VL, PCR products were obtained with the MKC primer incombination with the MKV1 and MKV2 primers. For 7C10 VH, PCR productswere obtained with the MHC-1 primer in combination with the MHV8 andMHV12 primers. A thorough sequencing of the PCR products cloned in thepGem-T easy vectors revealed two different sequences for the light chainand one unique sequence for the heavy chain.

a) Variable Region Isolated from the Oligo MKV1:

The DNA sequence obtained is characteristic of a variable region offunctional Ig. This novel sequence is therefore presumed to be thatcoding for 7C10 VL. The DNA (SEQ ID Nos. 48 and 50) and amino acid (SEQID NO: 49) sequences of the cDNA coding for 7C10 VL are represented inFIG. 14.

b) Variable Region Isolated from the Oligo MKV2:

The gene coding for this light chain comes from an aberrant mRNAtranscript which is present in all the standard fusion partners derivedfrom the original MOPC-21 tumor of which the mouse myeloma Sp2/Oag14,which was used in order to produce the 7C10 hybridoma, is part. Thissequence contains an aberrant recombination between the V and J genes(deletion of four nucleotide bases involving a change in the readingframe) and a mutation of the invariable cysteine in position 23 totyrosine. These changes suggest that this light chain would benonfunctional although nevertheless transcribed to messenger RNA. TheDNA sequence of this pseudo light chain is not shown.

c) Variable Region Isolated from the Oligos MHV8 and MHV12:

The DNA sequences obtained with these two oligos are identical, apartfrom the sequence encoded by the oligo itself. This sequence is a novelsequence coding for a functional heavy chain presumed to be that of themonoclonal antibody 7C10.The DNA (SEQ ID Nos. 51 and 53) and amino acid(SEQ ID NO: 52) sequences of the cDNA coding for 7C10 VH are representedin FIG. 15.

EXAMPLE 9 Construction of Chimeric Mouse-Man Genes

The chimeric antibody 7C10 was constructed so as to have the mouse 7C10regions VL and VH connected to the human constant regions kappa andgamma-1, respectively. Oligos were used in order to modify the 5′ and 3′ends of the sequences flanking the DNA coding for 7C10 VL and VH inorder to allow their cloning in vectors for expression in mammaliancells. These vectors use the strong promoter HCMV in order effectivelyto transcribe the heavy and light chains of the chimeric antibody7C10.On the other hand, these vectors likewise contain the replicationorigin of SV40 allowing an effective replication of the DNA and, as aconsequence, as a transitory expression of the proteins in cos cells.

EXAMPLE 10 Expression and Evaluation of the Recognition Activity of theIGF-I Receptor of the Chimeric Antibody 7C10

The two plasmids containing the DNA coding for the chimeric 7C10antibody were cotransfected in cos -7 cells (ATCC number CRL-1651) inorder to study the transitory expression of the recombinant antibody.After incubation for 72 hours, the culture medium was removed,centrifuged in order to eliminate the cell debris and analyzed by theELISA technique for the production of human IgG1 (see Example 16) andthe recognition of the receptor for IGF-I (see Example 17).

The ELISA tests for measurement of concentrations of human IgG1/Kappashowed that the expression of the chimeric antibody 7C10 in the cos -7cells was between 300 and 500 ng/mm, which is comparable to the valuesobtained with the majority of antibodies.

The ELISA tests for recognition of the receptor for IGF-I show that thechimeric antibody recognizes it specifically and with a good relativeavidity (see FIGS. 3A, 3B and 3C). This provides the functional proofthat the good VH and VL of the 7C10 antibody have been identified. Inaddition, this chimeric form of 7C10 appears as being an indispensabletool in the evaluation of the affinity of the humanized forms.

EXAMPLE 11 Molecular Modeling of the Variable Regions of the MouseAntibody 7C10

In order to assist and to refine the humanization process by “CDRgrafting”, a molecular model of the VL and VH regions of the mouseantibody 7C10 was constructed. The model is based on thecrystallographic structure of the heavy chain 1AY1 and of the lightchain 2PCP.

EXAMPLE 12 Process of Humanization by CDR Grafting of the VariableRegion of the Light Chain of the Antibody 7C10 (7C10 VL)

a) Comparison of the Amino Acid Sequence of 7C10 VL with all the knownMouse VL Sequences:

As a preliminary step to humanization by CDR grafting, the amino acidsequence of 7C10 VL was first compared with all the mouse VL sequencespresent in the databank of Kabat. 7C10 VL has thus been identified asbelonging to the subgroup II of the Kappa light chains as defined byKabat et al., (In Sequences of proteins of immunological interest(5^(th) edn.), NIH publication No. 91-3242, US Department of Health andHuman Services, Public Health Service, National Institutes of Health,Bethesda, 1991). The VL regions of monoclonal antibodies of mice havinga sequence identity ranging up to 95% have been identified (DRB1-4.3(SEQ ID NO: 55): 95% and C94-5B11′CL (SEQ ID NO: 56): 95%, see FIG. 17).In order to attempt to identify the out of the ordinary residues in the7C10 VL sequence, the amino acid sequence of 7C10 VL (SEQ ID NO: 54) wasaligned with the consensus sequence of the subgroup II of the mousekappa chains (SEQ ID NO: 57) as defined by Kabat (see FIG. 17).

In the Kabat position number 3, the valine (V) normally present in thesubgroup II of the Kappa light chains according to Kabat (71%) isreplaced by a leucine (L). A leucine in this position is not rare sinceit is found, for example, in DRB1-4.3 and C94-5B11′CL. According to themolecular model, this residue does not seem to play a particular role.Consequently, the conservation of this residue in the humanized formwill not be envisaged.

In the Kabat position number 7, the threonine (T) normally present inthe subgroup II of the Kappa light chains according to Kabat (66%) isreplaced by an iso-leucine (I). An isoleucine in this position isrelatively rare since it is only found 15 times among all the mouse VLsequences known and never among human VL sequences. The molecular modelshows that this residue (17) points toward the surface of the moleculebut does not contact the CDRs (the residue of a CDR which is the closestwould be the arginine in Kabat position number 42). In addition, it doesnot seem very probable that this residue 17 directly contacts theantigen. Consequently, the conservation of this residue in the humanizedform will not be envisaged, at any rate at first.

In the Kabat position number 77, the arginine (R) normally present inthe subgroup II of the Kappa light chains according to Kabat (95.5%) isreplaced by a serine (S). A serine in this position is not rare.

b) Comparison of the Amino Acid Sequence of 7C10 VL with all the knownHuman VL Sequences:

In order to identify the best human candidate for the “CDR grafting”,the Kappa VL region of human origin having the greatest homologypossible with 7C10 VL was sought. To this end, the amino acid sequenceof mouse kappa 7C10 VL was compared with all the human Kappa VLsequences present in the database of Kabat. Mouse 7C10 VL had thegreatest sequence homology with the human kappa VL regions of subgroupII as defined by Kabat et al., (1991). VH regions of monoclonalantibodies of human origin have been identified having a sequenceidentity ranging up to 75.9% (GM607 (SEQ ID NO: 58), see FIG. 18) overthe whole of the 112 amino acids composing the variable region. Agerminal line of human origin, DPK15/A19 (SEQ ID NO: 59), having asequence identity of 76% (see FIG. 18) was also identified, GM607(Klobeck et al., 1984). GM607 was therefore chosen as a human sequencereceptive of CDRs (according to the definition of Kabat) of mouse 7C10VL. By comparing the GM607 sequences with that of the consensus sequenceof the human subgroup II (SEQ ID NO: 60) (FIG. 18), no particularresidue in the framework regions (Rch) could be identified, indicatingby the same fact that GM607 was a good candidate for CDR grafting.

c) Humanized Versions of 7C10 VL:

The following stage in the humanization process consisted in joining theCDRs of mouse 7C10 VL to the framework regions (Rch) of the human lightchain selected, GM607 (Klobeck et al., 1984). At this stage of theprocess, the molecular model of the mouse Fv regions of 7C10 isparticularly useful in the choice of the mouse residues to be conservedas being able to play a role either in the maintenance of thetridimensional structure of the molecule (canonical structure of theCDRs, VH/VL interface, etc.) or in the binding to the antigen. In theRchs, each difference between the mouse (7C10 VL) and human (GM607)amino acids was examined scrupulously (see Table 6). In addition, theparticular residues in the mouse sequence 7C10 VL which were identified(see example 12.a) were taken into account if needed.

In the first version humanized by “CDR grafting” of 7C10 VL, human 1, asingle change in the framework regions (Rch) of GM607 was carried out.This change concerns the residue 2 (nomenclature of Kabat) situated inRch 1.This residue enters in effect into the composition of thecanonical structure of the CDR 1 of 7C10 VL and could therefore becritical for maintaining this loop in its good conformation. The valinepresent in this position in the mouse 7C10 VL sequence is thus conservedin this same position in the humanized form (see Table 6 and FIG. 19 forthe amino acid sequence (SEQ ID NO: 61) and FIG. 20 for the DNA sequence(SEQ ID Nos. 62 and 64) and the amino acid sequence comprising thepeptide signal (SEQ ID NO: 63).

In the second version humanized by “CDR grafting” of 7C10 VL, human 2,no change in the Rchs of the human light chain GM607 has been made. Allthe residues of the Rchs are thus of human origin including the residue2 which has therefore been mutated in order to replace the valinepresent in mouse 7C10 VL by an isoleucine found in this same position inthe human light chain GM607 (see Table 6 and FIG. 19 for the amino acidsequence (SEQ ID NO: 65) and FIG. 21 for the DNA sequence (SEQ ID Nos.66 and 68) and the amino acid sequence comprising the peptide signal(SEQ ID NO: 67)). This human form 2 is therefore totally humanized(apart from, of course, CDRs themselves) since all the residues of theRchs are those of the light chain of human origin, GM607.

TABLE 6 Alignment of the amino acid sequences leading to the design ofthe remodeled human 7C10 V_(L) regions Mouse Human light germinalRemodeled Remodeled FR or chain line GM human human Kabat # CDR 7C10DPK15/A19 607 7C10 1 7C10 2 Comments  1 1 FR1 D D D D D  2 2 | V* I* I*V* I* Cano L1 4(16) Vernier zone  3 3 | L V V V V  4 4 | M M M M MVernier zone  5 5 | T T T T T  6 6 | Q Q Q Q Q  7 7 | I S S S S  8 8 | PP P P P  9 9 | L L L L L  10 10 | S S S S S  11 11 | L L L L L  12 12 |P P P P P  13 13 | V V V V V  14 14 | S T T T T  15 15 | L P P P P  1616 | G G G G G  17 17 | D E E E E  18 18 | Q P P P P  19 19 | A A A A A 20 20 | S S S S S  21 21 | I I I I I  22 22 | S S S S S  23 23 FR1 C CC C C  24 24 CDR1 R R R R R  25 25 | S* S* S* S* S* Cano L1 4(16)  26 26| S S S S S  27 27 | Q Q Q Q Q  27A 28 | S S S S S  27B 29 | I* L* L* i*i* Cano L1 4(16)  27C 30 | V L L i I  27D 31 | H H H H H  27E 32 | S S SS S  28 33 | N N N N N  29 34 | G G G G G  30 35 | N Y Y n N  31 36 | TN N t T  32 37 | Y Y Y Y Y  33 38 | L* L* L* L* L* Cano L1 4(16)  34 39CDR1 Q D D q Q  35 40 FR2 W W W W W Vernier zone  36 41 | Y Y Y Y YVH/VL inter Vernier zone  37 42 | L L L L L  38 43 | Q Q Q Q Q VL/VHinter  39 44 | K K K K K  40 45 | P P P P P  41 46 | G G G G G  42 47 |Q Q Q Q Q  43 48 | S S S S S  44 49 | P P P P P VL/VH inter (+)  45 50 |K Q Q Q Q  46 51 | L L L L L VL/VH inter Vernier zone  47 52 | L L L L LVernier zone  48 53 | I I I I* I* Cano L2 1(7) Vernier zone  49 54 FR2 YY Y Y Y Vernier zone  50 55 CDR2 K L L k K  51 56 | v* G* G* v* v* CanoL2 1(7)  52 57 | S* S* S* S* S* Cano L2 1(7)  53 58 | N N N N N  54 59 |R R R R R  55 60 | L A A l L  56 61 CDR2 Y S S y Y  57 62 FR3 G G G G G 58 63 | V V V V V  59 64 | P P P P P  60 65 | D D D D D  61 66 | R R RR R  62 67 | F F F F F  63 68 | S S S S S  64 69 | G* G* G* G* G* CanoL2 1(7) Vernier zone  65 70 | S S S S S  66 71 | G G G G G Vernier zone 67 72 | S S S S S  68 73 | G G G G G Vernier zone  69 74 | T T T T TVernier zone  70 75 | D D D D D  71 76 | F* F* F* F* F* Cano L1 4(16)Vernier zone  72 77 | T T T T T  73 78 | L L L L L  74 79 | K K K K K 75 80 | I I I I I  76 81 | S S S S S  77 82 | S R R R R  78 83 | V V VV V  79 84 | E E E E E  80 85 | A A A A A  81 86 | E E E E E  82 87 | DD D D D  83 88 | L V V V V  84 89 | G G G G G  85 90 | V V V V V  86 91| Y Y Y Y Y  87 92 | Y Y Y Y Y VL/VH inter  88 93 FR3 C C C C C  89 94CDR3 F M M f F VL/VH inter  90 95 | Q* Q* Q* Q* Q* Cano L3 1(9)  91 96 |G A A g G VL/VH inter  92 97 | S L L s S  93 98 | H Q Q h H  94 99 | V TT v V  95 100 | P* P* P* P* P* Cano L3 1(9)  96 101 | W Q w W VL/VHinter (+)  97 102 CDR3 T T T T  98 103 FR4 F F F F VL/VH inter (+)Vernier zone  99 104 | G G G G 100 105 | G Q Q Q 101 106 | G G G G 102107 | T T T T 103 108 | K K K K 104 109 | L V V V 105 110 | E E E E 106111 | I I I I 107 112 FR4 K K K K Legend: The first column (Kabat)indicates the position of the amino acid residue according to Kabat etal., (1991); the second column (#) indicates the position of the aminoacid residue in the regular sequence; the third column (FR or CDR) wasmade in order easily to identify the segments of the skeleton (FR1, FR2,FR3 and FR4) and the CDR segments (CDR1, CDR2 and CDR3) (“CDR” for“Complementarity-Determining Region”) with the three CDRs separating thefour FRs; the fourth column (Mouse light chain 7C10) represents theamino acid sequence (SEQ ID NO: 54) of the V_(L) region of mouseantibody 7C10; the fifth column (Human germinal line DPK15/A19)represents the amino acid sequence (SEQ ID NO: 59) of the kappa II humanV light chain of the germinal line; the sixth column (GM607) representsthe amino acid sequence (SEQ ID NO: 58) of the V_(L) region of the humanantibody GM607; the seventh and eighth columns (remodeled human 7C10 1and 2) represent the amino acid sequences of the humanized 1 and 2antibody 7C10 VL (respectively SEQ ID Nos. 61 and 65). “*” indicates theparts of the canonical structure of the CDR loop such as defined byChothia et al., (Nature, 342, 877-883, 1989).

EXAMPLE 13 Process of Humanization by CDR Grafting of the VariableRegion of the Heavy Chain of the Antibody 7C10 (7C10 VH)

a) Comparison of the Amino Acid Sequence of 7C10 VH with all of theknown Mouse VH Sequences:

As a preliminary stage in humanization by CDR grafting, the amino acidsequence of 7C10 VH was first compared with all the mouse VH sequencespresent in the Kabat databank. 7C10 VH has thus been identified asbelonging to the subgroup I(A) of the heavy chains as defined by Kabatet al., (1991). VH regions of mouse monoclonal antibodies having asequence identity ranging up to 90.5% were identified (AN03′CL (SEQ IDNO: 70), see FIG. 22). In order to attempt to identify the out of theordinary residues in the sequence of 7C10 VH, we aligned the amino acidsequence of 7C10 VH (SEQ ID NO: 69) with the consensus sequence (SEQ IDNO: 71) of the subgroup I(A) of the mouse heavy chains as defined byKabat (see FIG. 22).

Residue 17 (Kabat's numbering), Thr for the consensus sequence ofsubgroup I(A) and Ser in 7C10 VH, is located on the surface of themolecule with respect to the interface with the constant region. Thisresidue does not seem to be important.

Residue 27 (Kabat's numbering), Asp for the consensus sequence ofsubgroup I(A) and Tyr in 7C10 VH, is a canonical residue for the CDR 1.Tyr in this position is not rare and is probably critical formaintaining CDR 1 in its good conformation.

Residue 84 (Kabat's numbering), Thr for the consensus sequence of thesubgroup I(A) and Asn in 7C10 VH. Asn was found 93 times in mouse VH and3 times in human VH. According to the molecular model, it is a surfaceresidue remote from the paratope.

The numbering of the amino acids is that of Kabat et al., (1991). Theresidues in the framework regions (apart from CDRs) which differ between7C10 VH and Kabat mouse subgroup I(A) are underlined. AN03′CL representsthe sequence of the heavy chain of a mouse antibody (access number inthe Kabat databank is P001289).

b) Comparison of the Amino Acid Sequence of 7C10 VH with all of theknown Human VH Sequences:

In order to identify the best human candidate for the “CDR grafting”,the VH region of human origin having the greatest possible homology with7C10 VH was sought. To this end, the amino acid sequence of mouse 7C10VH was compared with all the human VH sequences present in the Kabatdatabank. Mouse 7C10 VH had the greatest sequence homology with thehuman VH regions of the subgroup II as defined by Kabat et al., (1991).VH regions of monoclonal antibodies of human origin were identifiedhaving a sequence identity ranging up to 67.3% (human VH FUR1′CL (SEQ IDNO: 73, see FIG. 23) over the whole of the 98 amino acids encoded by thevariable gene (that is to say apart from CDR3 and region J). A germinalline of human origin, 4.22 VH IV (Sanz et al., 1989), having a sequenceidentity of 68.4%, according to the same criteria as for VH FUR1′CL, wasalso identified (human Germ-line (SEQ ID NO: 74), see FIG. 23). Thesequence encoded by the germinal line 4.22 VH IV was chosen as a humansequence receptive of the CDRs (according to the definition of Kabat) ofmouse 7C10 VH rather than VH FUR1′CL because in comparing the sequencesof 4.22 VH IV and VH FUR1′CL with that of the consensus sequence of thehuman subgroup II (human Kabat sg II (SEQ ID NO: 72), see FIG. 23 andtable 7), no atypical residue in the framework regions (Rch) could beidentified for 4.22 VH IV although the presence of two atypical residues(GIn and Arg in positions 81 and 82A according to the nomenclature ofKabat, respectively) were identified in the sequence encoded by VHFUR1′CL.

c) Humanized Versions of 7C10 VH:

The following stage in the humanization process consisted in joining theCDRs of mouse 7C10 VH to the framework regions (Rch) of the humangerminal line 4.22 VH IV (Sanz et al., 1989). At this stage of theprocess, the molecular model of the mouse Fv regions of 7C10 isparticularly useful in the choice of the mouse residues to be conservedas being able to play a role in the maintenance of the tridimensionalstructure of the molecule (canonical structure of the CDRs, VH/VLinterface, etc.) or in the binding to the antigen (belonging to theparatope). In the Rchs, each difference between the mouse (7C10 VH) andhuman (4.22 VH IV) amino acids was examined scrupulously (see Table 7).In addition, the particular residues in the mouse 7C10 VH sequence whichhad been identified (see Example 8.a) were taken into account if needed.

In the first version of 7C10 VH humanized by “CDR grafting”, humanized1, four changes in the framework regions (Rch) of 4.22 VH IV werecarried out (see Table 7, FIG. 24 for the amino acid sequence (SEQ IDNO: 75) and FIG. 25 for the DNA sequence (SEQ ID Nos. 76 and 78) and theamino acid sequence comprising the peptide signal (SEQ ID NO: 77)).These four changes concern:

-   -   Residue 30 (Kabat's nomenclature) situated in Rch 1. This        residue enters in effect into the structural composition of the        CDR1 of 7C10 VH (as defined by Chothia et al., 1989) and could        therefore be critical for maintaining this loop in its correct        conformation. The Thr present in this position in the mouse        sequence 7C10 VH is therefore conserved in this same position in        the humanized form.    -   Residue 48 (Kabat's nomenclature) situated in Rch 2. This        residue is close to the CDRs, although according to the        molecular model not in direct contact with the latter, and could        influence their ultimate conformation. The methionine present in        this position in the mouse sequence 7C10 VH is therefore        conserved in this same position in the humanized form 1.    -   Residue 67 (Kabat's nomenclature) situated in Rch 3. This        residue is close to the CDRs and according to the molecular        model could contact Lysine 60 (Kabat's nomenclature) in the        CDR 2. The isoleucine present in this position in mouse sequence        7C10 VH is therefore conserved in this position in the humanized        form 1.    -   Residue 71 (Kabat's nomenclature) situated in Rch 3. This        residue is part of the canonical structure of the CDR 2 and        should therefore be critical for maintaining this loop in its        correct conformation. The arginine present in this position in        the mouse sequence 7C10 VH is therefore conserved in this        position in the humanized form 1.

In the second version of 7C10 VH humanized by “CDR grafting”, humanized2, two changes in the framework regions (Rch) of 4.22 VH IV were carriedout. These two changes concern the residues 30 and 71 (Kabat'snomenclature), already described in the humanized form 1 (see Table 7,FIG. 24 for the amino acid sequence (SEQ ID NO: 79) and FIG. 26 for theDNA sequence (SEQ ID Nos. 80 and 82) and the amino acid sequencecomprising the peptide signal (SEQ ID NO: 81)).

In the third form of 7C10 VH humanized by “CDR grafting”, humanized 3,no change in the framework regions (Rch) of 4.22 VH IV was carried out.All the residues of the Rchs are therefore of human origin including theresidues 30, 48, 67 and 71 (Kabat's nomenclature) which have beenconserved (see Table 7, FIG. 24 for the amino acid sequence (SEQ ID NO:83) and FIG. 27 for the DNA sequence (SEQ ID Nos. 84 and 86) and theamino acid sequence comprising the peptide signal (SEQ ID NO: 85)). Thishumanized form 3 is therefore totally humanized (apart, of course, fromthe CDRs themselves as defined by Kabat) since all the residues of theRchs are those encoded by the VH gene of the germinal line 4.22 VH IV.

TABLE 7 Alignment of the amino acid sequences leading to the design ofthe remodeled human 7C10 V_(H) regions Mouse heavy Germinal HumanRemodeled Remodeled Remodeled FR or chain line FUR1′CL Human Human HumanKabat CDR 7C10 4.22 VH IV VH 7C10 H 1 7C10 H 2 7C10 H 3 Comments  1 FR1D Q Q Q Q Q  2 | V V V V V V Vernier Zone  3 | Q Q Q Q Q Q  4 | L L L LL L  5 | Q Q Q Q Q Q  6 | E E E E E E  7 | S S S S S S  8 | G G G G G G 9 | P P P P P P  10 | G G G G G G  11 | L L L L L L  12 | V V V V V V 13 | K K K K K K  14 | P P P P P P  15 | S S S S S S  16 | Q E E E E E 17 | S T T T T T  18 | L L L L L L  19 | S S S S S S  20 | L L L L L L 21 | T T T T T T  22 | C C C C C C  23 | S T T T T T  24 | V V V V* V*V* canonical H1 2(6)  25 | T S S S S S  26 | G* G* G* G* G* G* canonicalH1 2(6)  27 | Y* Y* Y* Y* Y* Y* canonical H1 2(6) Vernier Zone  28 | S SS S S S Vernier Zone  29 | I* I* I* I* I* I* canonical H1 2(6) VernierZone  30 FR1 T S S t T S Vernier Zone Close to the CDRs  31 CDR1 G S S gG q  32 | G G G G G G  33 | Y Y Y Y Y Y  34 | L Y Y I L I  35 | W* W* W*W* W* W* canonical H1 2(6) VH/VL interface  35A CDR1 N G G n N n  36 FR2W W W W W W  37 | I I I I I I VH/VL interface  38 | R R R R R R  39 | QQ Q Q Q Q VH/VL interface  40 | F P P P P P  41 | P P P P P P  42 | G GG G G G  43 | N K K K K K  44 | K G G G G G  45 | L L L L L L VH/VLinterface (+)  46 | E E E E E E  47 | W W W W W W VH/VL interfaceVernier Zone  48 | M I I m I I Vernier Zone Close to the CDRs  49 FR2 GG G G G G Vernier Zone  50 CDR2 Y S S y Y y Vernier Zone  51 | I I M I II  52 | S Y F s S s  53 | Y H H y Y y  54 | D S S d D d  55 | G* G* G*G* G* G* canonical H2 1(16)  56 | T S S t T t  57 | N T S n N n  58 | NY Y n N n  59 | Y Y Y Y Y Y  60 | K N N k K k  61 | P P P P P P  62 | SS S S S S  63 | L L L L L L  64 | K K K K K K  65 CDR2 D S S d d d  66FR3 R R R R R R  67 | I V V i V V Vernier Zone Close to the CDRs  68 | ST T T T T  69 | I I I I I I Vernier Zone  70 | T S S S S S  71 | R* V*V* r* r* V* canonical H2 1(16) Vernier Zone  72 | D D D D D D  73 | T TT T T T Vernier Zone  74 | S S S S S S  75 | K K K K K K  76 | N N N N NN  77 | Q Q Q Q Q Q  78 | F F F F F F Vernier Zone  79 | F S S S S S  80| L L L L L L  81 | K K Q K K K  82 | L L L L L L  82A | N S R S S S 82B | S S S S S S  82C | V V V V V V  83 | T T T T T T  84 | N A A A AA  85 | E A A A A A  86 | D D D D D D  87 | T T T T T T  88 | A A A A AA  89 | T V V V V V  90 | Y Y Y Y Y Y  91 | Y Y Y Y Y Y VH/VL interface 92 | C C C C C C  93 | A A A A A A VH/VL interface Vernier Zone  94 FR3R* R* R* R* R* R* canonical H1 2(6) Vernier Zone  95 CDR3 Y G y y yVH/VL interface  96 | G R g g g  97 | R Y r r r  98 | V C v v v  99 | FS f f f 100 | S 100A | T 100B | S 100C | C 100D | N 100E | W 100K | F Ff f f VH/VL interface (+) 101 | D D d d d 102 CDR3 Y P y y y 103 FR4 W WW W W VH/VL interface (+) Vernier Zone 104 | G G G G G 105 | Q Q Q Q Q106 | G G G G G 107 | T T T T T 108 | T L L L L 109 | L V V V V 110 | TT T T T 111 | V V V V V 112 | S S S S S 113 FR4 S S S S S Legend: Thefirst column (Kabat) indicates the position of the amino acid residueaccording to Kabat et al., (1991); the second column (FR or CDR) wasmade in order easily to identify the segments of the skeleton (FR1, FR2,FR3 and FR4) and the CDR segments (CDR1, CDR2 and CDR3) with the threeCDRs separating the four FRs; the third column (Mouse heavy chain 7C10)represents the amino acid sequence (SEQ ID NO: 69) of the V_(H) regionof the mouse antibody 7C10; the fourth column (Germinal line 4.22 VH IV)represents the amino acid sequence of the gene 4.22 VH IV (Sanz et al.,1989) (SEQ ID NO: 74); the fifth column (human FUR1′CL VH, kabataccession number N020619) represents the amino acid sequence (SEQ ID NO:73) [lacuna] IgMK antilamin B of human origin (Mariette et al., 1993);the sixth, seventh and eighth columns (remodeled human 7C10 1, 2 and 3)represent the amino acid sequences of the V_(H) region of remodeledhuman 7C10 respectively for the versions 1 (SEQ ID NO: 75), 2 (SEQ IDNO: 79) and 3 (SEQ ID NO: 83). “*” indicates the parts of the canonicalstructure of the CDR loop such as defined by Chothia et al., (1989).

EXAMPLE 14 Construction of the Genes Coding for the Humanized Versions 1of 7C10 VL and VH by Assembly of Oligonucleotides

a) Principle:

The genes (leader peptide+variable regions VDJ for VH or VJ for VK)coding for the humanized variable regions were synthesized bysolid-phase assembly on magnetic beads coated with streptavidin. Thegenes coding for humanized 7C10 VH (445 base pairs) and humanized 7C10VL (433 base pairs) are constructed by fusing two fragments of DNA owingto the presence of a Kpnl restriction site present in the two sequencesand situated almost halfway along the gene (at 200 and 245 nucleotideswith respect to the 5′ end of the gene for VL and VH, respectively). Thetwo fragments which are fused together are themselves assembled by anassembly technique which consists in using phosphorylatedoligonucleotides (approximately 30-35 mer) hybridized two by two (oneoligo sense and the other antisense, with a homology of approximately50%) in such a way that they overlap during elongation. A firstoligonucleotide biotinylated in the 5′ position is attached to themagnetic beads and then the pairs of phosphorylated oligonucleotides areadded one by one. The phosphodiester linkage between the juxtaposedphosphorylated oligonucleotides is produced by the enzyme T4 DNA ligase.

The genes thus synthesized de novo can be cloned directly (by digestionwith restriction enzymes compatible with the expression vector chosen)or amplified by PCR in order to obtain more material as a prelude todirectional cloning by enzymatic digestion. The sequence of the genethus constructed by de novo assembly is then verified by automaticsequencing of the DNA.

b) Experimental Protocol of the De Novo Assembly Technique:

Oligonucleotides phosphorylated in the 5′ position or biotinylated inthe 5′ position whose concentration was adjusted to 100 μM were orderedfrom MWG Biotech (see the sequences of the oligonucleotides used inTable 7 for the construction of humanized 7C10 VL, and Table 9 for theconstruction of humanized 7C10 VH). The oligonucleotides were hybridizedin pairs (an equimolar mixture, 500 pmol, of a sense oligo and of anantisense oligo in the buffer T4 DNA ligase is heated to 95° C. for 5minutes and then allowed to cool on the bench to ambient temperature)according to a scheme described in Table 10.

The first biotinylated oligonucleotide is attached to magnetic beadscoated with streptavidin (Dynabeads M-280 streptavidin, Dynal productNo. 112-05). For this, 500 pmol of the biotinylated oligonucleotide in a15 mM NaCl solution are added to 50 μl of the decanted beads (use of amagnet holder) previously washed twice with 100 μl of TE 1X buffer(Tris-EDTA 100× buffer: 1 M Tris-HCl, pH 8, 0.1 M EDTA, Sigma T-9285).After incubation at 37° C. for 15 min, the beads are washed twice withthe wash buffer (10 mM Tris-HCl pH 7.6, 10 mM EDTA and 50 mM NaCl) andthe pairs of hybridized oligo-nucleotides are then added one by one. Oneach readdition of a pair of oligonucleotides, the mixture is heated to95° C. for 5 min and then allowed to cool on the bench to ambienttemperature. Once ambient temperature is reached, 2 μl of 10 U/μl T4 DNAligase (Biolabs) are added and the mixture is incubated for 20 min at37° C. The beads are then washed (wash buffer) and the following pairsof oligonucleotides are then added in succession.

The last unpaired oligo (antisense) is assembled in the followingfashion. 5 μl of oligo (500 pmol) and 43 μl of T4 DNA ligase buffer areadded to the decanted beads, then the mixture is heated to 95° C. for 5min and allowed to cool on the bench to ambient temperature. Onceambient temperature is reached, 2 μl of T4 DNA ligase are added and themixture is incubated at 37° C. for 20 min. The beads are then washedtwice with wash buffer and then twice with TE 1× buffer.

The beads can then be conserved at 4° C. before proceeding to thecloning and sequencing of the gene assembled de novo.

TABLE 8 DNA sequence of oligonucleotides used for theconstruction of humanized 7C10 VL 1 by de novo assembly:LeaderMluI.biotin 5′-GTCAGAACGCGTGCCGCC (SEQ ID No. 87) 7C10Lresh.1sense5′-ACCATGAAGTTGCCTGTTAGGCTGTTGCTGCT (SEQ ID No. 88) 7C10Lresh.2sense5′-GATGTTCTGGTTTCCTGCTTCCGCAGTGATG (SEQ ID No. 89) 7C10Lresh.3sense5′-TTGTGATGACTCAGTCTCCACTCTCCCTGCCC (SEQ ID No. 90) 7C10Lresh.4sense5′-GTCACCCCTGGAGAGCCGGCCTCCATCTCCTG (SEQ ID No. 91) 7C10Lresh.5sense5′-CAGGTCTAGTCAGACCATTATACATAGTAATG (SEQ ID No. 92) 7C10Lrosh.6sense5′-GAAACACCTATTTGGAATGGTACCTGCAGA (SEQ ID No. 93) 7C10Lresh.7anti5′-GGCAACTTCATGGTGGCGGCACGCGTTCTGAC (SEQ ID No. 94) 7C10Lresh.8anti5′-GAAACCAGAACATCAGCACCAACAGCCTAACA (SEQ ID No. 95) 7C10Lresh.9anti5′-CTGAGTCATCACAACATCACTCCTGGAAGCAG (SEQ ID No. 96) 7C10Lresh.10anti5′-TCTCCAGGGGTGACGGGCAGGGAGAGTGGAG (SEQ ID No. 97) 7C10Lresh.11anti5′-TCTGACTAGACCTGCAGGAGATGGAGGCCGGC (SEQ ID No. 98) 7C10Lresh.12anti5′-AAATAGGTGTTTCCATTACTATGTACAATGC (SEQ ID No. 99) 7C10Lresh.13sense5′-CAGGGCAGTCTCCACAGCTCCTGATCTATAAA (SEQ ID No. 100) 7C10Lresh.14sense5′-GTTTCTAATCGGCTTTATGGGGTCCCTGACAG (SEQ ID No. 101) 7C10Lresh.15sense5′-GTTCAGTGGCAGTGGATCAGGCACAGATTTTA (SEQ ID No. 102) 7c10Lresh.16sense5′-CACTGAAAATCACCAGAGTGGAGGCTGAGGAT (SEQ ID No. 103) 7C10Lresh.17sense5′-GTTGGGGTTTATTACTGCTTTCAAGGTTCACA (SEQ ID No. 104) 7C10Lresh.18sense5′-TGTTCCGTGGACGTTCGGCCAAGGGACCAAGG (SEQ ID No. 105) 7C10Lresh.19sense5′-TGGAAATCAAACGTGAGTGGATCCTCTGCG (SEQ ID No. 106) 7C10Lresh.KpnIREV5′-TCTGCAGGTACCATTGC (SEQ ID No. 107) 7C10Lresh.KpnIbiotin5′-TGCAATGGTACCTGCAGAAGC (SEQ ID No. 108) 7C10Lresh.20anti5′-AGACTGCCCTGGCTTCTGCAGGTACCATTGCA (SEQ ID No. 109) 7C10Lresh.21anti5′-CGATTAGAAACTTTATAGATCAGGAGCTOTGG (SEQ ID No. 110) 7C10Lresh.22anti5′-TGCCACTGAACCTGTCAGGGACCOCATAAAGC (SEQ ID No. 111) 7C10Lresh.23anti5′-GATTTTCAGTCTAAAATCTGTGCCTGATCCAC (SEQ ID No. 112) 7C10Lresh.24anti5′-TAAACCCCAACATCCTCAGCCTCCACTCTGCT (SEQ ID No. 113) 7C10Lresh.25anti5′-TCCACGGAACATGTGAACCTTGAAAGCAGTAA (SEQ ID No. 114) 7C10Lresh.26anti5′-TTTGATTTCCACCTTGGTCCCTTGGCCGAAC (SEQ ID No. 115)7C10Lresh.BamHIantisense 5′-CGCAGAGGATCCACTCACG (SEQ ID No. 116)

TABLE 9 DNA sequence of oligonucleotides used for theconstruction of humanized 7C10 VH 1 by de novo assembly:LeaderMluI.biotin 5′-GTCAGAACGCGTGCCGCC (SEQ ID No. 117)7C10Lresh.1sense 5′-ACCATCAAAGTGTTGAGTCTGTTGTACCTCTTGA (SEQ ID No. 118)7C10Lresh.2sense 5′-CAGCCATTCCTGGTATCCTGTCTCAGGTGCAGCT (SEQ ID No. 119)7C10Lresh.3sense 5′-TCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCG (SEQ ID No. 120)7C10Lresh.4sense 5′-GAGACCCTGTCCCTCACCTGCACTGTCTCTGGT (SEQ ID No. 121)7C10Hresh.5sense 5′-TACTCCATCACCGGTGGTTATTTATGGAACTGG (SEQ ID No. 122)7C10Hresh.6sense 5′-ATACGGCAGCCCCCAGGGAAGGGACTGGAGTGG (SEQ ID No. 123)7C10Hresh.7sense 5′-ATGGGGTATATCAGCTACGACGGTACCAATAAC (SEQ ID No. 124)7C10Hresh.8antisense 5′-TCAACACTTTCATGGTGGCGGCACGCGTTCTGAC(SEQ ID No. 125) 7C10Hresh.9antisense5′-ATACCAGGAATGGCTGTCAAGAGGTACAACAGAC (SEQ ID No. 126)7C10Hresh.10antisense 5′-TGGGCCCGACTCCTGAAGCTGCACCTGAGACAGG(SEQ ID No. 127) 7C10Hresh.11antisense5′-TGAGGGACAGGGTCTCCGAAGGCTTCACCAGTCC (SEQ ID No. 128)7C10Hresh.12antisense 5′-CCACCGGTGATGGAGTAACCAGAGACAGTGCAGG(SEQ ID No. 129) 7C10Hresh.13antisense5′-CCCTGGGGGCTGCCGTATCCAGTTCCATAAATAA (SEQ ID No. 130)7C10Hresh.14antisense 5′-TAGCTGATATACCCCATCCACTCCAGTCCCTT(SEQ ID No. 131) 7C10Hresh.KpnIREV 5′-GTTATTGGTACCGTCG (SEQ ID No. 132)7C10Hresh.KpnIbiotin 5′-TACGACGGTACCAATAACTAC (SEQ 1D No. 133)7C10Hresh.15sense 5′-AAACCCTCCCTCAAGGATCGAATCACCATATC (SEQ ID No. 134)7C10Frosh.16sense 5′-ACGTGACACGTCCAAAGAACCAGTTCTCCCTGA (SEQ ID No. 135)7C10Hresh.17sense 5′-AGCTGAGCTCTGTGACCGCTGCGGACACTGCA (SEQ ID No. 136)7C10Hresh.18sense 5′-GTGTATTACTGTGCGAGATACGGTAGGGTCTT (SEQ ID No. 137)7010Hresh.19sense 5′-CTTTGACTACTGGGGCCAGGGAACCCTGGTCA (SEQ ID No. 138)7C10Hresh.20sense 5′-CCGTCTCCTCAGGTGAGTGGATCCTCTGCG (SEQ ID No. 139)7C10Hresh.21antisense 5′-AGGGAGGGTTTGTAGTTATTGGTACCGTCGTA(SEQ ID No. 140) 7C10Hresh.22antisense5′-ACGTGTCACGTGATATGGTGATTCGATCCTTG (SEQ ID No. 141)7C10Hresh.23antisense 5′-AGAGCTCAGCTTCAGGGAGAACTGGTTCTTGG(SEQ ID No. 142) 7C10Hresh.24antisense5′-CAGTAATACACTGCAGTGTCCGCAGCGGTCAC (SEQ ID No. 143)7C10Hresh.2Santisense 5′-AGTAGTCAAAGAAGACCCTACCGTATCTCGCA(SEQ ID No. 144) 7C10Hresh.26antisense5′-CTGAGGAGACGGTGACCAGGGTTCCCTGGCCCC (SEQ ID No. 145)7C10Hresh.BamHiantisense 5′-CGCAGAGGATCCACTCAC (SEQ ID No. 146)

TABLE 10 Oligonucleotide pairing protocol for the de novo assembly ofgenes coding for the humanized forms of 7C10 VH and VL: de novo assemblyde novo assembly of the MIUI-KpnI fragment of the KpnI-BamHI fragment of7C10 VL humanized 1 of 7C10 VL humanized 1 Biotinylated oligo leaderBiotinylated oligo 7C10 L MIUI 7C10 VL KpnI Oligo pair 1 and 7 Oligopair 13 and 20 Oligo pair 2 and 8 Oligo pair 14 and 21 Oligo pair 3 and9 Oligo pair 15 and 22 Oligo pair 4 and 10 Oligo pair 16 and 23 Oligopair 5 and 11 Oligo pair 17 and 24 Oligo pair 6 and 12 Oligo pair 18 and25 Antisense oligo 7C10 Oligo pair 19 and 26 VL KpnI Antisense oligo7C10 L BamHI Biotinylated oligo leader Biotinylated oligo 7C10 H MIUI7C10 VH KpnI Oligo pair 1 and 8 Oligo pair 15 and 21 Oligo pair 2 and 9Oligo pair 16 and 22 Oligo pair 3 and 10 Oligo pair 17 and 23 Oligo pair4 and 11 Oligo pair 18 and 24 Oligo pair 5 and 12 Oligo pair 19 and 25Oligo pair 6 and 13 Oligo pair 20 and 26 Oligo pair 7 and 14 Antisenseoligo 7C10 VH Antisense oligo 7C10 BamHI VH KpnI

EXAMPLE 15 Construction of the Genes Coding for the Humanized Versions 2of 7C10 VL and 7C10 VH and 3 of 7C10 VH by Directed Mutagenesis

The humanized version 2 of 7C10 VH was obtained by directed mutagenesisof the residues 48 and 67 (according to Kabat's nomenclature) ofversion 1. This directed mutagenesis was carried out with the aid of thesystem QuikChange™ Site-directed mutagenesis of Stratagene (kit #200518)according to the protocol described by the manufacturer. Theconstruction is carried out in two stages, first the residue 48 onversion 1 was mutated with the aid of the pair of primers7C10Hhumanized1QCM48 sense and antisense (see Table 11) and subsequentlythis version mutated at the residue 48 was itself mutated at the residue67 with the aid of the pair of primers 7C10Hhumanized1QC167 sense andantisense (see Table 11).

The humanized version 3 of 7C10 VH was obtained by site-directedmutation of the residues 30 and 71 (according to Kabat's nomenclature)of version 2 likewise using the system QuikChange™. This construction iscarried out in two stages. At first, the residue 30 on version 2 wasmutated with the aid of the primers 7C10HhumanizedQCT30 sense andantisense (see Table 11). Subsequently, this version mutated at theresidue 30 was itself mutated at the residue 71 by using the pair ofprimers 7C10Hhumanized1V67QCR71 sense and antisense (see Table 11).

The humanized version 2 of 7C10 VL was obtained by site-directedmutation of the residue 2 (according to Kabat's nomenclature) of version1 by using the system QuikChange™. The residue 2 on version 1 wasmutated by using the pair of primers 7C10Lhumanized1QCV2 sense andantisense (see Table 11).

TABLE 11 List of the oligonucleotides used for the directedmutagenesis by the stratagene QuikChange™ system: 7C10HhumanizedlQCT30.5′-CTGGTTACTCCATCAGCGGTGGTTATTTATC (SEQ ID No. 147) sense7C10HhumanizedlQCT30. 5′-CATAAATAACCACCGCTGATGGAGTAACCAG(SEQ ID No. 148) antisense 7C10HhumanizedlQCM48.5′-GCGACTGCACTGGATCGGGTATATCAGCTAC (SEQ ID No. 149) sense7C10HhumanizedlQCM48. 5′-GTAGCTGATATACCCGATCCACTCCAGTCCC(SEQ ID No. 150) antisense 7C10HhumanizedlQCI67.5′-TCCCTCAAGGATCGAGTCACCATATCACGTG (SEQ ID No. 151) sense7C10HhumanizedlQCI67. 5′-CACCTGATATGGTGACTCGATCCTTGAGGGA(SEQ ID No. 152) antisense 7C10HhumanizedlV67QCR71.5′-GATCGAGTCACCATATCAGTGGACACGTCCAAGAA (SEQ ID No. 153) sense CCAG7C10HhumanizedlV67QCR71. 5′-CTGGTTCTTGGACGTGTCCACTGATATGGTGACTC(SEQ ID No. 154) antisense GATC 7C10hhumanizedlQCV2.5′-GCTTCCAGCAGTGATATTGTGATGACTCAGT (SEQ ID No. 155) sense7C10LhumanizedlQCV2. 5′-ACTCAGTCATCACAATATCACTGCTGGAAGC (SEQ ID No. 156)antisense

EXAMPLE 16 Transfection of the cos7 Cells by Electroporation

The mammalian expression vectors containing the chimeric or humanizedversions of the heavy and light chains of the antibody 7C10 were testedin cos7 cells for the transitory expression of the recombinantantibodies 7C10. The DNA was introduced into the cos cells byelectroporation with the aid of a BioRad instrument (Gene Pulsar). TheDNA (10 μg of each vector) is added to aliquots of 0.8 ml of cos cellsat a concentration of 1×10⁷ cells per ml in PBS buffer (without Ca++ andMg++). A pulsation of 1900 volts and a capacity of 25 μF was delivered.The transfected cos cells are then added to 8 ml of DMEM mediumcontaining 5% of calf serum and incubated at 37° C. for 72 hours. Thesupernatant is then collected, centrifuged in order to eliminate thecell debris and tested by ELISA for the measurement of its concentrationof recombinant antibody 7C10 of IgG1/human Kappa type.

EXAMPLE 17 ELISA Method for Measuring the Concentrations of RecombinantAntibody IgG1/Human Kappa Present in the Supernatant of the cosTransfectants

The supernatants produced by transitory expression in cos7 cells weretested for the presence of 7C10 antibody of IgG1/human Kappa type.

For the detection of the IgG1/human Kappa immunoglobulin, 96-well ELISAplates (Maxisorb, Nunc) were coated with a goat anti-human IgGpolyclonal antibody (specific for the gamma Fc fragment, JacksonImmuno-Research Laboratories Inc., #109-005-098). The supernatants ofcos cells were diluted in series and added to the coated wells. Afterincubation for one hour at 37° C. and washing, a goat anti-human lightKappa chain polyclonal antibody conjugated to peroxidase (HRP, Sigma,A-7164) was added. After incubation for 45 minutes at 37° C. andwashing, the TMB substrate (KPL #50-76-04) was added. After incubationfor 10 minutes, the reaction was stopped by the addition of 1 M sulfuricacid and the optical density was read at 450 nm. A purified humanIgG1/human Kappa immunoglobulin (Sigma, 1-3889) of known concentrationwas used as a standard reference antibody.

EXAMPLE 18 ELISA Method for Determining the Recognition Aactivity of7C10 Recombinant Antibodies of Human IgG1/Kappa Type on the Receptor forIGF-I (IGF-IR)

The cos7 culture supernatants were tested for their capacity torecognize IGF-I R by an ELISA method. 96-well ELISA plates (DynexImmulon 2HB) were coated with 100 μl per well of a solution of PBScontaining 0.31 ng/μl of IGF-I R (Human Insulin-Like Growth Factor Isoluble Receptor, R & D Systems, #391-GR) by incubation for one night at4° C. After washing with PBS containing 0.05% Tween 20, the plates weresaturated by the addition of a solution of PBS containing 0.5% gelatinsolution and incubation at 37° C. for 1 hour. After three washes withPBS, the samples of cos supernatants to be tested, previously diluted inseries in PBS containing 0.1% gelatin and 0.05% Tween 20, were added tothe plates. After incubation at 37° C. for 1 hour followed by threewashes (PBS containing 0.05% Tween 20), an anti-human IgG antibody(specific for the Fc fragment) conjugated to peroxidase (HRP, JacksonImmuno-Research Laboratories Inc., #109-035-098) was added (dilution to1/5000 in PBS containing 0.1% gelatin and 0.05% Tween 20). Afterincubation for 45 minutes at 37° C. and 3 washes (PBS containing 0.05%Tween 20), the TMB substrate (KPL #50-76-04) was added. After incubationfor 10 minutes, the reaction was stopped by addition of 1 M sulfuricacid and the optical density was read at 450 nm.

EXAMPLE 19 Determination of the Recognition Activity of IGF1-R byDifferent Versions of the Humanized 7C10 Antibody by “CDR Grafting”

At first, we compared the recognition activity of humanized forms 1 ofthe heavy and light chains of 7C10 for the IGF-I receptor with respectto the chimeric form. FIG. 28 shows the results of an ELISA test ofrecognition of the IGF-IR (see Example 18) from supernatants of the cos7cells whose concentration of IgG1/human Kappa had been previouslydetermined by ELISA (see Example 17). The titration curves of the fourrecombinant antibodies tested overlap perfectly indicating that theirrelative affinities for IGF-IR are very similar. It is thereforeconcluded from this that the humanized form 1 of 7C10, composed of thehumanized light chain 1 (1 single mouse residue present in the frameworkregions) in combination with the humanized heavy chain 1 (4 mouseresidues present in the framework regions), specifically recognizes theIGF-I receptor and has an affinity very similar to that of the chimericantibody (mouse variable regions).

Subsequently, we looked at the influence of the residue 2 (according toKabat's nomenclature) of the humanized light chain of 7C10 (humanizedversion 1 versus humanized 2, see FIG. 19) on the recognition of theIGF-IR. FIG. 29 shows the results of the ELISA test for recognition ofthe IGF-IR (see Example 18) from supernatants of cos7 cells whoseconcentration of IgG1/human Kappa had been previously determined byELISA (see Example 17). The two humanized versions 1 and 2 of the lightchain had been combined successively with humanized 7C10 VH 1. Thetitration curves of the two combinations are superimposed indicatingthat the mutation of residue 2 of the light chain, which has beenchanged from one valine in the humanized version 1 to an isoleucine inthe humanized form 2, apparently has no influence on the relativeaffinity of recognition of the IGF1 receptor. The humanized form 2 ofthe light chain of 7C10 thus forms one version where no mouse residue(apart from CDRs) has been conserved. This version, totally humanized,represents the preferred version of 7C10 VL.

The totally humanized version of the 7C10 light chain (humanized version2, see above) was tested in combination with the three humanizedversions of the heavy chain of 7C10. FIG. 30 shows the results of theELISA test for recognition of the IGF-IR from supernatants of cos7 cellswhose concentration of IgG1/human Kappa had been previously determinedby ELISA (see Example 17). The titration curves are very similar andvirtually overlap with the reference curve corresponding to the chimericantibody, indicating that the three humanized versions 1, 2 and 3 of7C10 VH give an identical relative affinity for IGF-IR when they arecombined with humanized 7C10 VL 2. Other ELISA tests conducted inparallel (results not shown) have however revealed that a point mutationof the residue 71 (Kabat's nomenclature) from an arginine (mouse) to avaline (human) involved a small loss of affinity of the correspondingantibody for IGF-IR. It is thus reasonable to think that humanized 7C10VH 2 has the same relative affinity for IGF-IR as humanized 7C10 VH 1.This humanized form 2 will therefore be preferred with respect to theform 1 since it only has two mouse amino acids (residues 30 and 71, seeFIG. 24). The humanized form 3 which does not have any mouse residue(apart from CDRs) will also be preferred since it only seems to involvea minimal loss of affinity.

In conclusion, it appears that two humanized forms of the antibody 7C10according to the present invention are particularly preferred. A formconstituted by the combination of humanized 7C10 VH 2 (2 conserved mouseresidues) with humanized 7C10 VL 2 (no conserved mouse residue) andanother form constituted by the combination of humanized 7C10 VH 3 (noconserved mouse residue) with humanized 7C10 VL 2 (no conserved mouseresidue). This last form constitutes the ultimate humanized versionsince no mouse residue is present at the same time in the heavy andlight chains.

EXAMPLE 20 Expression of EGFR and of IGF-IR on the Surface of A549 Cells

The synergy of action obtained by the coadministration of two MABsdirected respectively against IGF-IR and EGFR was studied in nude micecarrying a non-small cell lung tumor established by subcutaneousinjection (s.c.) of A549 cells (lung carcinoma cell line).

At first, and in order to ensure the presence of the two receptorsIGF-IR and EGFR on the surface of the A549 cell before injecting thisinto the mouse, labeling for FACS reading of these cells was carried outwith, respectively, the murine 7C10 anti-IGF-IR MAB (FIG. 37B) and themurine 225 anti-EGFR MAB (FIG. 37D). In order to do this, the cells weresaturated for 30 min at 4° C. with a solution of PBS 10% FCS (fetal calfserum), washed and then incubated for 30 min at 4° C. with the MAB ofinterest. After 3 new washes, the secondary anti-species antibodycoupled to FITC (fluorescein isothiocyanate) is added. After incubationfor 30 min, reading on the FACS (Fluorescence Activated Cells Sorter) iscarried out at 520 nm (excitation 488 nm).

The results presented in FIGS. 37A to 37D show that the A549 cells haveon their surface a comparable number of receptors for EGF and IGF1. Inthe two cases, the population is homogeneous with respect to thedistribution of each of the receptors. The specificity of the labelingis confirmed by the use of an isotype control (FIG. 37C). These resultsvalidate the use of the A549 cell as a model for the study of a synergyof action on two IGF-IR and EGFR receptors and for the study of acollaboration of these two receptors.

EXAMPLE 21 Synergy of Action of an Anti-IGF-IR MAB and of an Anti-EGFRMAB Coadministered In Vivo, in the Nude Mouse in the Context of anAntitumor Treatment

For this study, nude mice are grafted s.c. with 5.10⁶ A549 cells. Fivedays after the cell graft, the tumors are measured and a homogeneousbatch of mice in terms of tumor volume is formed. Starting from thisbatch, groups of 6 mice are generated at random. These mice will betreated intraperitoneally (i.p.), twice per week with each of the MAB7C10 and 225 individually at the dose of 250 μg/mouse or with the twoMAB in coadministration. The MAB 9G4 is administered as an experimentisotype control.

The results presented in FIG. 38 show that each of the antibodies 7C10and 225 administered alone is capable of inducing a significant decreasein the tumor growth in vivo. It can be noted that the two MAB testedhave a comparable activity on the growth of the tumor A549. In asurprising fashion with respect to the literature, a significant synergyis observed during simultaneous administration of the two MAB (p<or=0.01 at each of the times of the kinetics in a t-test) suggesting thata collaboration of the two receptors exists for the optimum growth of atumor in vivo and that, contrary to the data in the literature, theblockage of one of the two axes does not suffice to totally inhibit thegrowth mediated by the second.

EXAMPLE 22 Study of the Antitumor Activity of the Murine Antibodies 7C10and 225 Coadministered in Mice Orthotopically Implanted with A549 Cells

The use of orthotopic models for the evaluation of the antitumoractivity presents a particular interest with respect to the process ofmetastatic dissemination of a tumor. In order to evaluate the antitumoractivity of an antibody mixture directed respectively against IGF-IR andEGFR, 10⁶ A549 cells (non-small cell lung cancer) were implanted in theintrapleural cavity of nude mice. It is to be noted that the consequenceof this type of tumor implantation is a metastatic dissemination similarto that observed in man and leads to the death of the animals. FIG. 39shows that the administration of the antibodies 225 and 7C10 aloneallows a comparable and a significant gain in survival to be observed.In a surprising fashion, the coadministration of these two antibodiesincreases in a considerable fashion the survival of the animalssuggesting that this treatment could have an impact on the metastaticdissemination of the tumor cells.

EXAMPLE 23 7C10 and 7H2HM Inhibit the Phosphorylation of the Tyrosine ofthe β Chain of IGF-IR and of IRS-I

MCF7 cells are cultured for 24 hours at 5.10⁴ cells/cm² (75 cm² plates,COSTAR) in 20 ml of RPMI without phenol red, mixed with 5 mM ofglutamine, penicillin/ streptomycin (respectively 100 U/100 μg/ml) and10% of fetal calf serum. After three washes in PBS, the cells wereincubated for 12 hours in medium (RPMI) without phenol red, devoid offetal calf serum and mixed with 5 mM of glutamine,penicillin/streptomycin, bovine serum albumin at 0.5 μg/ml (SigmaA-8022) and transferrin at 5 μg/ml (Sigma T8158).

For activation, the cells were first incubated at 37° C. for 2 minuteswith blocking antibodies (10 μg/ml) and then IGF-I (Sigma I3769, 50ng/ml) was added for two additional minutes. The reaction was stopped byaspiration of the incubation medium and the plates were laid on ice. Thecells were solubilized by addition of 0.5 ml of lysis buffer (50 mMtris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate),mixed with protease inhibitors (1 tablet per 50 ml, Boehringer Ref.:1697 498), and phosphatase inhibitors (Calbiochem Ref.: 524625 (1/100)).The cells were scraped off and the suspension was recovered and placedon a shaker at 4° C. for 1.5 hours. The solutions were centrifuged at12,000 rpm for ten minutes (4° C.) and the protein concentrations of thesupernatants were quantified by BCA.

500 μg of proteins of the cell lysate were mixed with the anti-IGF-IR(Santa cruz Ref.: sc-713) for immunoprecipitation and incubated on theshaker at 4° C. for 1.5 hours. The immunoprecipitates were recovered byaddition of protein A-agarose (Boehringer Ref.: 1 134 515) and incubatedall night on the shaker at 4° C. For the immunoprecipitation of IRS-1,anti-IRS-1 antibodies coupled to agarose beads (Santa cruz Ref.: 559Ac)were used. The agarose beads were washed twice with 1 ml of lysisbuffer, twice with a wash buffer 1 (50 mM tris-HCl pH 7.5; 500 mM NaCl;0.1% Nonidet P40; 0.05% sodium deoxycholate (Boehringer 1 332 597),mixed with protease inhibitors and phosphatase inhibitors) and once witha wash buffer 2 (50 mM tris-HCl; 0.1% Nonidet P40; 0.05% sodiumdeoxycholate (Boehringer Ref.: 1 332 597), mixed with proteaseinhibitors and phosphatase inhibitors 1/100). The immunoprecipitateswere resuspended in a Laemmli buffer, heated to 100° C. for 5 minutes.The supernatants were analyzed by electrophoresis on polyacrylamide SDSgel (8% Novex EC6015). The proteins were transferred to a nitrocellulosemembrane followed by either an immunoblot with anti-phosphotyrosineantibodies conjugated to HRP (upstate Biotechnology 4G10) or betaanti-chain of IGF-IR or anti-IRS-1 (Santa Cruz Ref.: sc 8038) followedby an anti-rabbit antibody conjugated to HRP. The imprints were revealedby chemiluminescence (Amersham RPN 2209) followed by autoradiography onKodak X-mat AR films.

FIG. 40A represents MCF7 cells nonstimulated (0) or stimulated eitherwith IGF-I (50 ng/ml) alone (0+IGF-I) or combined with monoclonal orhumanized anti-IGF-IR antibodies (10 μg/ml) 7C10, 1H7, 7H2HM. Theantibodies 9G4 or hIgG1 are murine or human immunoglobulins of isotypeIgG1 used as an experiment negative control. The beta chains of theIGF-IR were immunoprecipitated and blotted with phosphorylatedanti-tyrosine antibodies. The results obtained show that the monoclonalor humanized anti-IGF-IR 7C10, 1 H7 and 7H2HM antibodies inhibit thephosphorylation of the tyrosine of the beta chain of the IGF-IR.

FIG. 40B represents MCF7 cells nonstimulated (0) or stimulated eitherwith IGF-I (50 ng/ml) alone (0+IGF-I) or combined with monoclonal orhumanized anti-IGF-IR antibodies (10 μg/ml) 7C10, 1H7, 7H2HM. Asdescribed above, the antibodies 9G4 or hIgG1 are murine or humanimmunoglobulins of isotype IgG1 used as an experiment negative control.The IRS-1 was immunoprecipitated and blotted with phosphorylatedanti-tyrosine antibodies. The results obtained show that the monoclonalantibodies 7C10, 7H2HM and 1 H7 inhibit the phosphorylation of thetyrosine of the IRS-1.

EXAMPLE 24 7C10 and 7H2HM Induces the Internalization of the IGF-IR

MCF7 and A549 cells were suspended to 1.10⁷ cells/ml in PBS with 10% offetal calf serum (FACS buffer). 1.10⁶ cells were incubated for 30minutes at 37° C. with the monoclonal antibodies at 10 μg/ml (7C10, 7G3,9G4) or at 20 μg/ml for 7H2HM. After washing, the cells were labeled at4° C. for 30 minutes with a biotinylated anti-IGF-IR (monoclonalantibody 12B1) and finally incubated at 4° C. for 30 minutes with aconjugate of streptavidin-488 alexa Fluor®. The cells were analyzed byFACScan (Becton-Dickinson, Enembogegem, Belgium) with the Cellquestsoftware after elimination of debris.

FIG. 41 shows the A549 cells without coloration (1^(st) peak), the A549cells incubated with 7C10 or 7H2HM (2^(nd) peak) and the A549 cellsincubated with an irrelevant mouse or rat IgG1 (3^(rd) peak). A decreaseby two of the surface expression of the IGF-IR by the cells is seen whenthe cells have been previously incubated with 7C10 or 7H2HM.

EXAMPLE 25 7C10 and 7H2HM Induce the Degradation of the IGF-IR

MCF-7 cells were cultured for 24 hours at 10.10⁴ cells/cm² (75 cm²,Costar) in 15 ml of complete medium. Next, the cultures were washedthree times with PBS and incubated for 12 hours with medium devoid ofserum. Next, the cells were incubated with cycloheximide at 25 μg/mlalone or with 10 μg/ml of monoclonal antibody 7C10, 9G4, 7G3 or of IGF-I(50 ng/ml). In certain experiments, before incubation with themonoclonal antibodies, the cells were treated for 1 hour at 37° C. withMG-132 (10 μM, Calbiochem 474791) in order to inhibit the proteasomeactivities. After incubation, the cells were washed and solubilized byaddition of a lysis buffer. 20 μg of proteins were analyzed byelectrophoresis on polyacrylamide gel at 8% of SDS and transferred to anitrocellulose membrane followed by a beta anti-chain immunoblot of theIGF-IR such as described further above.

The analysis by Western-blot (FIG. 42A) of the integrity of the IGF-IRshows that 7C10 and 7H2HM induce the degradation of the receptor whilethe natural ligand does not cause any degradation of the latter. Nodegradation of the receptor is observed with the 9G4, an irrelevantantibody used as an isotype control. FIG. 42B demonstrates, and withrespect thereto, that the degradation is inhibited by a proteasomeinhibitor MG132 (incubation period of 2 hours).

Comparable results were obtained with the humanized antibody 7H2HM (FIG.42C).

Additional experiments using the murin 7C10 Mab have been performed anddemonstrate that the degradation of IGF-IR is strongly proteasomedependant as the effect of 7C10 is totally abolished in presence of theproteasome inhibitor MG115 (FIG. 57A). This property seems to beparticular the our antibody as for other anti-IGF-IR described in theliterature or in published patents the down-regulation does not occurvia the proteasomal pathway (Sachdev et al., Cancer Res. 63, 627-635.2003; Burtrum et al., Cancer Res. 63, 8912-8921. 2003; patent US2004/0202655 A1 (Pharmacia)). In addition to this degradation pathway,the 7C10 MAb seems to also induce the degradation of IGF-IR via thelysosomal/endosomal pathways as a substantial reduction of downregulation was observed when cells are treated with either methylamineor chloroquine (FIGS. 57B and C)

After down-regulation the level of receptor remain significantly low atleast for 31 hours (FIG. 57D).

EXAMPLE 26 Evaluation of 7C10 and h7C10 Ability to Bind to IGF-IR andHybrid-R

Example 26.1: Evaluation of 7C10 and h7C10 ability to immunoprecipitateIGF-IR and hybrid-R purified from transfected cells respectively withIGF-IR and IR-A or IGF-IR and IR-B (thereafter referred as R+/IR-A orR+/IR-B

The goal of this study is to evaluate the ability of 7C10 and h7C10 toimmunoprecipitate IGF-IR, IR or Hybrid-R. 7C10 and h7C10 are compared to17-69 (which recognizes both IGF-IR well and Hybrid-R).

Method:

The used cells for this study are listed thereafter:

-   -   R+: R− fibroblasts stably transfected with the IGF-1 receptor        (IGF-IR) cDNA    -   R−/IR-A: R− fibroblasts stably transfected with the insulin        receptor isoform A (IR-A) cDNA    -   R−/IR-B:R− fibroblasts stably transfected with the insulin        receptor isoform B (IR-B) cDNA    -   R+/IR-A: R− fibroblasts stably co-transfected with the IGF-I and        the insulin receptor isoform A cDNA and, therefore, expressing        hybrid receptors A (Hybrid-RsA)    -   R+/IR-B: R− fibroblasts stably co-transfected with the IGF-I and        the insulin receptor isoform B cDNA and, therefore, expressing        hybrid receptors B (Hybrid-RsB)

For the obtention of cellular lysat, cells were solubilized in RIPAbuffer and 4 mg protein used for immunoprecipitation.

Cell lysates were immuprecipitated as follows:

-   R+with either 7C10 or h7C10-   R+/IR-A and R+/IR-B with either 7C10 or h7C10 or 17-69-   R−/IR-A and R−/IR-B with either MA-20 (an anti-IR antibody) or 7C10    or h7C10

Following immunoprecipitation, the pellet was resuspended in 2× samplebuffer and subjected to SDS-PAGE (7.5% polyacrylamide).

Filters were blotted as follows: Filters containing R+lysates (andtherefore only IGF-IR) with an anti-IGF-IR β-subunit (Santa Cruz).Filters containing lysates from all the remaining cells with an antibodyanti-IR β-subunit (Santa Cruz).

Results:

-   Two independent experiments are shown (FIG. 43A and FIG. 43B)

Comments:

1) 7C10 and h7C10 are equally efficient in immunoprecipitating theIGF-IR (lanes 1 and 2)

2) Neither 7C10 nor h7C10 appreciably immunoprecipitate IR

3) Both 7C10 and h7C10 recognizes Hybrid-R.

EXAMPLE 26-2 Displacement Analysis of IGF1 on IGF-IR by 7C10, h7C10 and1H7

IGF-IR from R+ cell lysates were immunocaptured in Maxisorb platescoated with 17-69 antibody.

¹²⁵I-IGF1 (FIG. 44) was then allowed to bind to immunocaptured receptorsin the absence or the presence of increasing concentrations of unlabeledligand (IGF1 or IGF2) or antibodies (7C10, h7C10, 1 H7, 9G4). Resultsare plotted as percent of maximal specific binding.

Both 7C10 and h7C10 displace labeled IGF1 with a very similar efficiencywith IC₅₀ values less than 100 nM, and in this example with IC₅₀ ofabout 1.3 nM and 1.9 nM respectively. By comparison, 1 H7 was much lesseffective with an IC₅₀ value up to 100 nM (FIG. 44).

EXAMPLE 26-3 Displacement Analysis of IGF1 on Hybrid-RsA by 7C10, h7C10and 1H7

Hybrid-RsA from R+/IR-A cell lysates were immunocaptured in Maxisorbplates coated with anti IR antibody 83-7.

125I-IGF1 (FIG. 45) was then allowed to bind to immunocaptured receptorsin the absence or the presence of increasing concentrations of unlabeledligand (IGF1 or IGF2) or antibodies (7C10, h7C10, 1 H7, 9G4). Resultsare plotted as percent of maximal specific binding.

Both 7C10 and h7C10 displace labeled IGF1 with a very similar efficiencywith IC₅₀ values less than 100 nM, and in this example of about 2.0 nMfor each. By comparison, 1 H7 was much less effective with an IC₅₀ valueup to 50 nM, preferably up to 100 nM (FIG. 45).

EXAMPLE 26-4 Displacement Analysis of IGF1 on Hybrid-RsB by 7C10, h7C10and 1H7

Hybrid-RsB from R−/IR-B cell lysates were immunocaptured in Maxisorbplates coated with 83-7 antibody.

125I-IGF1 (FIG. 46) was then allowed to bind to immunocaptured receptorsin the absence or the presence of increasing concentrations of IGF1 andIGF2 or antibodies (7C10, h7C10, 1 H7, 9G4). Results are plotted aspercent of maximal binding.

Both 7C10 and h7C10 displace labeled IGF1 with a very similar efficiencywith IC50 values less than 100 nM, and in this example of about 1.5 and2.5 respectively. By comparison, 1 H7 was much less effective with anIC50 value up to 100 nM (FIG. 46).

EXAMPLE 27 Internalization and Degradation Studies of the IGF-IR

Internalization and degradation studies were analyzed by FACS andwestern-blot analysis. Internalization studies were performed by FACSanalysis using a murine biotinylated anti-IGF-IR monoclonal antibody(Mab) thereafter described as 12B1 MAb and binding to an epitopedifferent from the one recognized by 7C10 and h7C10 antibodies. The 7G3MAb, a non neutralizing anti-IGF-IR was introduced as negative control.Both antibodies were generated in our laboratory. Confluent MCF-7 cellswere trypsinized and 1×10⁶ cells from each cellular suspension wasplated in 96-well plates in FACS buffer. Plates were incubated, eitherwith or without 25 μg/ml of cycloheximide (Calbiochem), 30 min at 37° C.with either IGF1 (50 ng/ml) or with 10 μg/ml of 7C10, 7G3, h7C10, mIgG1,hIgG1. Cells incubated with FACS buffer alone were used to determine thebasal level of expression of the IGF-IR. Then cells were washed twiceand 12 μg/ml of biotinylated-12B1 MAb were added to the plate. After 30min of incubation at 4° C. to avoid receptor internalization, cells werewashed 3 times at 4° C. and stained by addition of a streptavidin AlexaFluor® 488conjugate (Molecular Probes Europe BV, Leiden, Netherlands).

Both 7C10 and h7C10 cause a rapid down regulation of the IGF-IR with amaximum after 4 hours of incubation with the antibodies (Table 12). Nodown regulation was observed when cells were incubated either with IGF1,7G3 non neutralizing Mab, murine (mIgG1) or human (hIgG1) isotypecontrol. The absence of internalization when cells were incubated withIGF-I is probably due to the rapid recycling of IGF-IR; indeed thisrapid recycling phenomenon is well known by the man skill in the art forthis type of receptor. These results were observed either in presence orin absence of cyclohexemide. Observed results are shown in the followingTable 12.

TABLE 12 Cells incubated without Cyclohexemide Cells incubated withCyclohexemide mIgG1 12B1 mIgG1 12B1 Buffer Biotinylé Biotinylé BufferBiotinylé Biotinylé 5 min Buffer 8 8 135 8 8 90 IGF1 8 9 137 8 9 93 1 hBuffer 9 9 153 8 8 89 hIG1 8 9 150 8 9 92 h7C10 9 9 64 8 8 37 mIgG1 8 9144 8 8 88 7C10 9 9 61 8 9 36 7G3 8 9 137 8 8 85 4 h Buffer 8 8 136 8 895 hIgG1 8 8 139 7 8 94 h7C10 8 8 39 8 8 29 mIgG1 9 9 130 8 8 78 7C10 88 37 8 8 27 7G 8 8 109 8 8 72 16 h Buffer 8 9 135 8 9 85 HIgG1 9 9 144 88 85 H7C10 9 10 34 8 9 26 MIgG1 9 10 100 10 10 56 7C10 9 9 31 9 9 25 7G39 9 90 9 9 57

Table 12: Study of Antibody Induced IGF-IR Internalization by FACSAnalysis:

For immnunoblotting experiments 7.5×10⁶ cells were plated in 75 cm²flasks in 15 ml of complete medium (red phenol-free RPMI and Ham-F12Krespectively for MCF-7 and A549 both supplemented with 10% FCS and 1%L-Glutamine). Twenty four hours after plating, cells were washed 3 timeswith PBS and incubated for 24 additional hours at 37° C. Then medium wasremoved and cells incubated either 1 h, 4 h or 16 h at 37° C. with 15 mlof serum-free medium with or without antibodies to be tested or withIGF-I. Cells were then harvested and lysed in Tris HCl buffer pH 7.5,15% NaCl 1M (Sigma), 10% detergent mix (10 mM Tris-HCl, 10% Igepal)(Sigma), 5% sodium deoxycholate (Sigma), 1 protease inhibitor cocktailcomplete TM tablet (Roche) and 1% phosphatase inhibitor Cocktail Set 11(Calbiochem). For Western blot analysis, equal amount of cell lysateswere separated on 10% SDS-PAGE, transferred to nitrocellulose filters,probed with an anti-β IGF-IR rabbit polyclonal IgG (Santa Cruz Biotech),revelated with an anti rabbit IgG coupled to the HRP (AmershamBioscience) and visualized by ECL (Amersham Bioscience).

FIGS. 47A and 47B represent the study of antibody induced degradation ofthe IGF-IR.

For immnuno-blotting analysis (FIGS. 47A and 47B), experiments were donewithout cyclohexemide as the above experiment shows that no differencewas observed in presence or in absence of this compound. 7C10 and h7C10cause a comparable internalization of the IGF-IR in both A549 (A) andMCF-7 (B) cells. In MCF-7 cells the maximal internalization was observedafter four hours incubation with 7C10 and h7C10, whereas, for A549 themaximal internalization is observed as earlier as 1 hour. No degradationwas observed when cells were incubated either with IGF-I, 7G3 or murine(mIgG1) or human (hIgG1) isotype control.

EXAMPLE 28 Study of the Degradation Pathway of IGF- IR

7.5×10⁶ MCF-7 cells were plated in 75 cm² flasks in 15 ml of completemedium (red phenol-free RPMI supplemented with 10% FCS and 1%L-Glutamine). Twenty four hours after plating, cells were washed 3 timeswith PBS and incubated for 24 additional hours at 37° C. in 15 mlserum-free medium . Then medium was removed and cells incubated for twohours in 7.5 ml of serum-free medium either containing 30 μM MG115 orDMSO. Then, 7.5 ml of serum-free medium with or without h7C10, hIgG1 orIGF-I were added for 4 additional hours. Cells were then harvested andlysed in Tris HCl buffer pH 7.5, 15% NaCl 1M (Sigma), 10% detergent mix(10 mM Tris-HCl, 10% Igepal) (Sigma), 5% sodium deoxycholate (Sigma), 1protease inhibitor cocktail complete TM tablet (Roche) and 1%phosphatase inhibitor Cocktail Set II (Calbiochem). For Western blotanalysis, equal amount of cell lysates were separated on 10% SDS-PAGE,transferred to nitrocellulose filters, probed with an anti-β IGF-IRrabbit polyclonal IgG (Santa Cruz Biotech), revelated with an antirabbit IgG coupled to the HRP (Amersham Bioscience) and visualized byECL (Amersham Bioscience). FIG. 48 shows the obtained results.

To further characterize the pathway of degration of the h7C10 antibody,cells were incubated 4 hours with either IGF-I or human isotype control(hIgG1) in presence or in absence of the proteasome inhibitor MG115. Inthe herein described experiment h7C10 induced, a dramatic degradation ofthe IGF-IR either in presence or in absence of DMSO. No degradation wasobserved when IGF-I or hIgG1 were added. When cells were incubated with30 μM MG115, no down regulation of the IGF-IR was observed demonstratingthat the down regulation of IGF-IR on MCF-7 observed in FIG. 2 occursthrough the proteasome pathway. This property is surprising and ofparticular interest. Indeed none of the anti-IGF-IR antibody alreadydescribed for inducing a degradation of the IGF-IR (Malauney E K and al,Cancer Research, 2003; Sachdev D and al, Cancer Research, 2003) involvedthe proteasome pathway for degradation.

Actually, it has been reported that IGF-IR is internalized and degradedvia a lysosome-dependent pathway (Alessi et al., B. Curr. Biol., 1997).In addition, both Mab391 (Hailey et al., Molecular Cancer Therapeutics,2002) and scFv-Fc (Sachdev et al., Cancer Research, 2003) down regulateIGF-IR by the endocytic pathway.

As a consequence, regarding the present knowledge, it can not be excludethat h7C10 also down regulate, in addition to the proteasome pathway aspreviously described, via other known and described pathways foranti-IGF-IR antibodies, i.e., lisosome-dependent and/or endocyticpathways.

Such a property, if validated, is of particular interest as it woulddemonstrate the capacity of the h7C10 to interact with differentsignalization/degradation pathways, and thus its therapeutic efficacy.Supplementary studies are in progress in order to validate thishypothesis.

EXAMPLE 29 Anti-Tumoral Activity of the Murine Antibody 7C10Co-Administrated with an Anti-VEGF Antibody on Mice OrthopicallyImplanted with A549 Cells

One million of A549 NSCLC were implanted through the chest wall into theleft pleural cavity space of 6 weeks old Swiss nude mice following theprotocol described by Klaus-Berthier et al., (Kraus-Berthier, L., Jan,M., Guilbaud, N., Naze, M., Pierre, A., and Atassi, G. Histology andsensitivity to anticancer drugs of two human non-small cell lungcarcinomas implanted in the pleural cavity of nude mice. Clin. CancerRes. 6 (1): 297-304, 2000). Seven days after the cell injection, micewere treated i.p. with a loading dose of 250 μg of antibodies, and them,twice a week with 125 μg of antibodies. For the combined therapy,antibodies were mixed prior to the injection.

The anti-VEGF antibody used was an IgG2b, clone 26503.11 commercializedby SIGMA. It was described as a neutralizing antibody (Ferrara N. etal., Biochem. Res. Com. 161:851. 1999; Ferrara et al., Endocrinol.Revieuw 13:18.1992; Leung D.W. et al., Science 246 :1306.1989).

FIG. 48 shows that a combined therapy increase dramatically the timesurvival compared to untreated mice or to mice treated with singletherapy.

The T/C % are calculated according the following formula, [MEDIAN OFTREATED MICE/MEDIAN OF CONTROL MICE×100]. The obtained T/C % are about134% and 144% for the 7C10 and anti-VEF antibody respectively. For thecombined treatment 7C10 +anti-VEGF antibodies, the T/C % is 188%.

As a conclusion, similarly to the co-administration of 7C10+225 (seeexample 22), the co-administration of 7C10 +anti-VEGF antibodiesincrease the mice survival.

EXAMPLE 30 Production of Deoxyvinblastine

4′-R deoxyvinblastine (structure see below Scheme 1) is obtained byionic reduction of anhydrovinblastine according to a process known tothose skilled in the art (Lafitte C et al., Tetrahedron Letters, 1998,Volume 39, pp. 8281-8282).

4′-S deoxyvinblastine, or 4′-S deoxyleurosidine, is obtained bycatalytic hydrogenation of anhydrovinblastine according to the techniquealso known to those skilled in the art (De-Bruyn A. et al., Bulletin ofthe Belgian Chemical Society, 1983, Volume 92, number 5, pp 485-494).

EXAMPLE 31 Deacetylation of Vinca Dimeric Alkaloids

Deoxyvinblastine or deoxyleurosidine is dissolved and stirred for 4hours at 50° C. in 30 ml of methanol containing 1.2 equivalents ofsodium methoxide. This solution is then poured into ice-cold water inorder to precipitate the compound formed. After filtration, washing withwater and drying under vacuum at 40° C., 4-deacetyldeoxyvinblastine or4-deacetyldeoxyleurosidine is obtained, with a purity of greater than95%.

EXAMPLE 32 Direct Coupling of 4′-deoxyvinblastine (4′R) or4′-deoxyleurosidine (4′S) by Reaction of a 4-carboxyhydrazide Functionon the Pre-Oxidized Anti-IGF-IR Antibodies

The 4′-deoxyvinblastine or the 4′-deoxyleurosidine is treated withanhydrous hydrazine in solution in methanol and at ambient temperature.The reaction is monitored by Analytical High Performance LiquidChromatography (HPLC) and, when 95% of the starting alkaloid hasreacted, the reaction medium is treated with water in order for the4′-deoxyvinblastine-3-deacetyl-4-carbohydrazide or the4′-deoxyleurosidine-3-deacetyl-4-carbohydrazide to be separated byfiltration.

After silica gel chromatography and then crystallization, the4′-deoxyvinblastine-3-deacetyl-4-carbohydrazide or the4′-deoxyleurosidine-3-deacetyl-4-carbohydrazide is greater than 96%pure.

The anti-IGF-IR antibody is oxidized under cold conditions in a sodiumacetate buffer by treatment with sodium meta-periodate. After exclusionchromatography, the oxidized anti-IGF-IR antibody, in solution in anacetate buffer, is treated under cold conditions with the4′-deoxyvinblastine-3-deacetyl-4-carbohydrazide or the4′-deoxyleurosidine-3-deacetyl-4-carbohydrazide.

The immunoconjugate thus obtained is separated from the unconjugatedresidual Vinca alkaloid and purified by exclusion chromatography with aphosphate buffer at pH 7.4, and then intensive dialysis. The absence offree Vinca alkaloid is verified by analytical HPLC.

The immunoconjugate is characterized on an SDS PAGE-type electrophoresisgel (Coomassie blue and/or silver nitrate), by exclusion chromatography(SEC, UV at 280 nm) and by MALDI-TOF mass spectrometry. The mapping ofthe coupling sites is carried out by means of analysis by liquidchromatography coupled to mass spectrometry (LC MS), subsequent toenzyme digestion (trypsin and PNGase F) (Laguzza et al., J. MED. CHEM.,1989, 32:548).

EXAMPLE 33 Coupling of the 4′-deoxyvinblastine (4′R) or the4′-deoxyleurosidine (4′S) to the Anti-IGF-IR Antibodies by Virtue ofSuccinic Anhydride

The 3-deacetyl-4′-deoxyvinblastine or the 3-deacetyl-4′-deoxyleurosidineis treated with succinic anhydride in pyridine for 24 hours at 20° C.The reaction is monitored by analytical HPLC and, when 95% of thestarting alkaloid has reacted, the reaction medium is treated with waterin order to precipitate the 3-deacetyl-4′-deoxyvinblastine hemisuccinateor the 3-deacetyl-4′-deoxyleurosidine hemisuccinate. After filtrationand drying, the compound is purified by reverse-phase preparative HPLCusing C18 grafted silica and an eluent made up of acetonitrile, methanoland ammonium acetate buffer.

The 3-deacetyl-4′-deoxyvinblastine hemisuccinate or the3-deacetyl-4′-deoxyleurosidine hemisuccinate is treated withhydroxybenzotriazole and dicyclohexylcarbodiimide in dimethylformamideat ambient temperature for 24 hours and in the presence of a catalyticamount of dimethylaminopyridine. After filtration, the solution is mixedwith the anti-IGF-IR monoclonal antibody at pH 8.6 for 4 hours. Theimmunoconjugate is separated from the unconjugated Vinca alkaloid byexclusion chromatography with a phosphate buffer at pH 7.4. Intensivedialysis makes it possible to eliminate the unconjugated Vinca alkaloid.The immunoconjugate is characterized by SDS PAGE gel electrophoresis, byexclusion chromatography and by MALDI TOF mass spectrometry. The mappingof the coupling sites is carried out by means of liquid chromatographyanalysis coupled to mass spectrometry (LC MS), subsequent to enzyme(trypsin) digestion, compared to a reference tryptic map obtained forthe non-derived monoclonal antibody (Schneck et al., Clin. Pharmacol.Ther., 1990, 47:36; Rowland et al., Cancer. Immunol. Immunother., 1985,19:1).

EXAMPLE 34 Coupling of the 4′-deoxyvinblastine (4′R) or the4′-deoxyleurosidine (4′S) on a Nitrogen-Containing Residue of theAnti-IGF-IR Antibodies by Virtue of a Disulfide Bridge Included in theLinkage

The 3-deacetyl-4′-deoxyvinblastine or the 3-deacetyl-4′-deoxyleurosidineis treated, in methylene chloride, at ambient temperature for 24 hours,in the presence of a catalytic amount of dimethylaminopyridine, with alarge excess of 3-methyldisulfanylpropanoic acid and a large excess ofdicyclohexylcarbodiimide. The reaction medium is treated conventionallyand the 3-deacetyl-4′-deoxyvinblastine 3-methyldisulfanylpropanoate orthe 3-deacetyl-4′-deoxyleurosidine 3-methyldisulfanylpropanoate is thenpurified by reverse-phase preparative HPLC using C18 grafted silica andan eluent made up of acetonitrile, methanol and ammonium acetate buffer.

The 3-deacetyl-4′-deoxyvinblastine 3-methyldisulfanylpropanoate or the3-deacetyl-4′-deoxyleurosidine 3-methyldisulfanylpropanoate is treatedwith dithiothreitol in a mixture of water and methanol so as to obtain3-deacetyl-4′-deoxyvinblastine 3-sulfanylpropanoate or3-deacetyl-4′-deoxyleurosidine 3-sulfanylpropanoate, which is purifiedby reverse-phase preparative HPLC using C18 grafted silica and an eluentmade up of acetonitrile, methanol and ammonium acetate buffer.

The anti-IGF-IR antibody is derivatized with N-succinimidyl4-(2-pyridyldithio)propanoate (the trade name of which is SPDP) in a 50mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl and 2 mMEDTA, for 90 minutes. Added to this solution of antibody thusderivatized is the 3-deacetyl-4′-deoxyvinblastine 3-sulfanylpropanoateor the 3-deacetyl-4′-deoxyleurosidine 3-sulfanylpropanoate dissolved ina minimum of DMSO. After contact for 24 hours, the immunoconjugate isisolated by exclusion chromatography and is characterized on an SDS PAGEelectrophoresis gel, by exclusion chromatography and by MALDI TOF massspectrometry (Ojima et al., J. Med. Chem., 2002, 45:5320).

EXAMPLE 35 Coupling of the 4′-deoxyvinblastine (4′R) or the4′-deoxyleurosidine (4′S) to the Aanti-IGF-IR Antibodies by Virtue of aTerminal Hydrazide Function Carried by a Linkage Connected to the VincaAlkaloid

The 3-deacetyl-4′-deoxyvinblastine or the 3-deacetyl-4′-deoxyleurosidineis treated, in methylene chloride at ambient temperature for 24 hours,in the presence of a catalytic amount of dimethylaminopyridine, with anexcess of methyl monoester of 1,6-hexanedicarboxylic acid and an excessof dicyclohexylcarbodiimide. The reaction medium is treatedconventionally and the 3-deacetyl-4′-deoxyvinblastine 3-methoxycarbonylpentanoate or the 3-deacetyl-4′-deoxyleurosidine 3-methoxycarbonylpentanoate is then purified by reverse-phase preparative HPLC using C18grafted silica and an eluent made up of acetonitrile, methanol andammonium acetate buffer.

The 3-deacetyl-4′-deoxyvinblastine 3-methoxycarbonyl pentanoate or the3-deacetyl-4′-deoxyleurosidine 3-methoxycarbonyl pentanoate is treatedby default with anhydrous hydrazine in solution in methanol at ambienttemperature. The reaction is monitored by analytical HPLC and, when 70%of the starting alkaloid has reacted, the reaction medium is evaporatedand the 3-deacetyl-4′-deoxyvinblastine 3-hydrazinocarbonyl pentanoate orthe 3-deacetyl-4′-deoxyleurosidine 3-hydrazinocarbonyl pentanoate ispurified by reverse-phase preparative HPLC using C18 grafted silica andan eluent made up of acetonitrile, methanol and ammonium acetate buffer.

The oxidation of the anti-IGF-IR antibody, the coupling with3-deacetyl-4′-deoxyvinblastine 3-hydrazinocarbonyl pentanoate or3-deacetyl-4′-deoxyleurosidine 3-hydrazinocarbonyl pentanoate, thepurification and the identification are carried out according to thesame techniques as those described in Example 32.

EXAMPLE 36 Activity, Compared In Vivo, of the 7C10 and h7C10 Antibodieson the A549 and MCF-7 Models

In order to confirm the activity of the humanized antibody h7C10 invivo, the latter was compared with 7C10 in the MCF-7 oestrogen-dependentbreast tumor model and in the A549 non-small-cell lung tumor model.

To do this, 5.10⁶ A549 cells were implanted subcutaneously in nude mice.Five days after this implantation, the tumors were measured and groupsof 6 mice were formed. These groups were treated, respectively, with 1)the 7C10 antibody injected ip (intraperitoneally) at a rate of 125μg/dose twice a week; 2) the h7C10 antibody injected under the sameconditions as its murine form; 3) PBS (it has been shown previously thatmurine and human control isotypes do not modify the tumor growth profilecompared to treatment of the animals with PBS). In the MCF-7 breasttumor model, a sustained-release oestradiol granule (0.72 mg/tabletreleased over 60 days) is implanted subcutaneously 24 hours beforeimplantation of the cells. This granule is essential to theestablishment of any E2-dependent human tumor in this animal species.

FIGS. 50 and 51 show, as expected, that significant inhibition of tumorgrowth is observed with the 7C10 murine antibody. As regards the h7C10humanized antibody, the activity observed is of exactly the sameintensity as that observed with its murine counterpart, whatever themodel used. This datum indicates that the humanization has not modifiedthe properties of the antibody generated.

EXAMPLE 37 Demonstration of the Compared Activities of Vinblastine, ofVincristine, of 4′S Deoxyvinblastine and of 4′R Deoxyleurosidine

The greater activity of the (4′R) deoxyvinblastine and of the (4′S)deoxyleurosidine was demonstrated in vivo against intravenously-graftedP388 murine leukemia and compared with the activity of vinblastine andof vincristine tested under the same conditions. The protocol for thistest is described by Kruczynski A. et al., Cancer Chemotherapy andPharmacology, 1998, volume 41, pages 437 to 447.

To do this, a total of 10⁶ P388 murine leukemia cells were implantedi.v. in CDF1 mice on day 0. After randomization of the animals in cagesfor treatment with each alkaloid and control cages, the compounds wereadministered i.p. on day 1.

Conventionally, the in vivo activity of compounds is expressed by theincrease in survival time. The survival time is expressed by the T/C ata dose expressed in mg per kg (mg/kg). The T/C corresponds to the ratio,multiplied by 100, of the median of the survival time of the treatedanimals to the median of the survival time of the control animals. Inagreement with the standard criteria of the NCl, a T/C of 120corresponds to a minimum level for concluding that activity is present.

A T/C of between 120 and 175 makes it possible to conclude that there issignificant activity and a T/C above 175 makes it possible to concludethat there is a high level of anti-leukemia activity. A T/C below 75expresses toxicity of the test compound at the dose administered.

Table 13 below gives the results obtained with a minimum of 7 and amaximum of 15 treated mice for each group of animals treated with aVinca alkaloid or for the control group.

Table 13 gives the results of T/C values obtained for each Vincaalkaloid tested.

FIGS. 52 and 53 show the greater anti-leukemia activity of the 4′R and4′s deoxyvinblastines compared to vinblastine and vincristine.

TABLE 13 Dose in mg/kg 0.63 1.25 2.5 5 10 20 40 T/C for vinblastine 114114 129 143 57 T/C for vincristine 114 143 143 100 57 T/C for 4′-S- 114143 200 100 57 deoxyvinblastine T/C for 4′-R- 100 100 129 143 200 214 43deoxyvinblastine

EXAMPLE 38 Demonstration of the In Vivo Antitumour Activity of 4′ R- and4′ S-deoxyvinblastine Conjugated with IGR-IR Antibodies on Human Tumorsof Various Origins

In order to demonstrate the benefit of addressing the chemotherapycompounds (4′R) and (4′S) deoxyvinblastine (respectively called RDV andSDV in FIG. 5) with a humanized antibody directed against IGF-IR, 5.10⁶A549 non-small-cell lung cancer cells were implanted in a subcutaneousposition on the right flank of Swiss Nude mice. Seven days afterimplantation of the cells, the tumors can be measured and the animalsare distributed randomly into 6 groups of 6 mice and treated accordingto the following protocol:

-   -   h7C10: twice a week at a rate of 250 μg/dose throughout the        entire duration of the experiment;    -   RDV and SDV: 4 intraperitoneal injections 7 days apart at the        dose of 0.35 mg/kg, which corresponds to the dose of each of the        compounds present in the conjugates;    -   the groups of animals given the chemotherapy compounds coupled        to the antibody receive respectively 0.35 mg/kg of each of the        chemotherapy agents and 250 μg/dose of antibodies. These        conjugates are administered according to the same modes as the        groups given the chemotherapy compounds alone;    -   the animals of the control batch are given injections of PBS,        administered according to the same frequency.

The weight of the mice and the tumor volume are evaluated twice a week.The tumor volumes are calculated according to the formula: ½(length.width.height).

The results are shown in FIG. 53.

The animals given only RDV or SDV evolve in the same manner as thecontrol group, which seems coherent with respect to the optimum dosesusually injected for these two compounds, which are respectively 20mg/kg and 2.5 mg/kg. Surprisingly, when each of the compounds is coupledto the h7C10 antibody, a very significant inhibition of the tumor growthis observed. This inhibition is significantly greater than that observedwith the antibody alone, administered at the same concentration.

All these results appear to indicate that targeting of the cells withthe h7C10 antibody promotes concentration of the drug in the cell to betargeted and makes it possible to observe, as a result, significantinhibitions of tumor proliferation at low doses of chemotherapy product,and in particular at doses which are completely non-toxic in mice, as isdemonstrated by the lack of weight loss of the animals (data notcommunicated).

EXAMPLE 39 In Vivo Down-Regulation of IGF-IR

To confirm if previous in vitro observations (Examples 24 and 25) ofdown-regulation of IGF-IR levels by 7C10 and h7C10 was one of themechanisms responsible for inhibition of tumor growth in vivo, theeffect of h7C10 on IGF-IR levels in mice with MCF-7 xenograft tumors wastested. Nine mice bearing a xenograft tumor were studied. Tumors wereresected from 3 mice before treatment (described as T0 in FIG. 59). Then3 mice received i.p. injections of 1 mg of A2CHM and 3 other micereceived i.p. injections of a hIgG1 used as isotype control. Six hoursafter treatment, mice were sacrificed and tumors harvested. Tumorsamples were frozen in liquid nitrogen and homogenized in a lysisbuffer. A total of 50 μg of tumor extracts immunoblotted for totalIGF-IRb levels. In this experiment, he cytokeratin 19 (CK19) wasimmunoblotted with an antibody recognizing specifically the CK19 fromMCF-7 cells as control of the amount of tumor protein loaded in eachlane. FIG. 59 shows that the amount of protein loaded is comparable inall lanes excepted in lane 9 which could not be considered asinterpretable. All MCF-7 xenograft tumors had high levels of IGF-IR innon treated mice (lanes 1-3). Six hours after treatment, tumor takenfrom animals treated with h7C10 had significant down regulation ofIGF-IR (lanes 4-6). In two mice which received the isotype control nochanges in the level of IGF-IR were observed within tumor tissue (lanes7 and 8). These results confirm that the down-regulation of IGF-IR isone of the mechanisms involved in the in vivo activity of h7C10.

EXAMPLE 40 Cell Lines with IGF-IR Overexpressed and/or Abnormally OverActivated

The following cell lines are known as overexpressing and/or displayingover activated IGF-IR:

-   -   Prostate (PC3, DU145),    -   breast (MCF-7, T47D, BT20, ZR-75-1, MDA-MB-231,    -   lung (A549, A427, SK-LU-1)    -   colon (HT29, Co1o205, CaCo-2),    -   thyroid (BC-PAP, FRO, ARO),    -   ovarian (SK-OV-3),    -   pancreas (BxPC3, MiaPaCa-2, LN36),    -   renal, adrenal cancer, sarcomas (SK-ES-1), medulloblastoma        (Daoy, TE-671, D283 Med),    -   retinoblastoma, multiple myeloma (MM-1 S, MM-1 R), melanoma        (SK-MEL-28)

The level of expression of some of the described cells above can bedetermined as follow:

To determine the level of expression of IGF-IR on various cell lines,cells were labeled with 5 μg/ml of 7C10 antibody. 9G4 was used asirrelevant isotype control. Anti-mouse MAb coupled to FITC was used forrevelation of the bound 7C10. In parallel, beads from DAKO QIFIKITbearing quantified amounts of CD5 receptors were used for standard curvedetermination. After FACS analysis, beads yield a MFI. A standard curverelating MFI and receptor number was determined. MFI measured after cellstaining with 7C10 was reported on the standard curve and receptornumber on each tumor cell line was determined.

Quantification of the number of IGF-IR on tumor cell line surface showsthat all the cell lines analyzed express between 1200 to 96000receptors/cell.

Some preferred results are:

IGF-IR (MFI ± sd) JURKAT  1201 ± 833 PC3  3903 ± 1178 CAKI 1 15887 ± ndT29 17102 ± 2210 A431 18530 ± 2126 LnCAP 19738 ± 1080 COLO205 23355 ±5711 BxPC3 25185 ± 3463 A549 32179 ± 5418 CAKI 2 42673 ± 693 MCF-7 96072± 2807

EXAMPLE 41 Cell Lines Expressing Hybrid-R

The following cell lines are known as overexpressing and/or displayingover activated hybrid-R and/or IGF-IR (Siddle et al., Harm. Res. 41:56-65, 1994; Lynn Seely et al., Endocrinology. 136: 1635-1641, 1995 andPandini et al., Clinical Cancer Res. 5: 1935-1944. 1999):

-   -   Breast cancer (MDA-MB-231, MDA-MB-157, MDA-MB-468, MDA-MB-453,        ZR-75),    -   thyroid cancer (BC-PAP)

EXAMPLE 42

To determine the variation of IGF-IR between normal and tumor tissues,paraffin embedded section of either breast or lung cancer have beenstained with a biotinylated-conjugated anti-IGF-IR monoclonal antibody.The result shown in FIG. 58 demonstrate that samples from patients witheither lung or breast cancer display significantly higher levels ofIGF-IR

EXAMPLE 43 Tumoral Markers:

TABLE 14 Markers Normal Values Associated Cancers ACE <5 ng/ml (for nonColorectal, pancreatic, smokers) stomach, breast, lung, <10 ng/ml (forthyroide smokers) aFP <15 ng/ml liver, testis βHCG <0.1 ng/ml testis,placenta (during pregnancy) CA125 <35 U/ml ovarian CA15.3 <30 U/mlbreast CA19.9 <37 U/ml pancreas, digestive system CA 72.4 <6 U/mlstomach, ovarian CYFRA 21.1 <3.3 ng/ml Non small cells lung cancer5-HIAA urinary 5 to 45 mmol/24 h carcinoïdes (digestive system) NSE<12.5 ng/ml Small cells lung cancer, central nervous system(neuroblastoma) SCC <1.5 ng/ml uterine, head and neck Total PSA: <4ng/ml prostate free PSA: 10 à 40% of total PSA TG (thyroglobuline) <3μg/l Thyroid CT (Calcitonine) <10 ng/l Thyroid LDH <2 N osteosarcoma(lacticodeshydro- genase)

The following non limitative references are incorporated by referenceand are describing several preferred markers.

-   -   PSA: Anaes, Service des recommendations professionnelles et        service evaluation economique, September 2004;    -   ACE: Fletcher 1986, Carcinoembryonic antigen. Ann. Intern. Med.,        104(1): 66-73/American Society 1996, J. Clin. Oncol. 14(10):        2843-77    -   CA19.9: Narimatsu et al., 1998, Cancer Res., 58(3)        512-8/Vestergaard et al., 1999, Clin. Chem., 45(1): 54-61    -   CA15.3: Hayes et al., 1986, J. Clin. Oncol., 4(10): 1542-50 /        Safi et al., 1989, Int. J. Biol. Markers, 4(4): 207-14/Devine et        al., 1995, Breast Cancer Res. Treat., 34(3): 245-51/Yasasever et        al., 1994, Eur. J. Gynaecol. Oncol., 15(1): 33-6    -   TG: National Academy 2001,

Piechaczyk et al., 1989, Clin. Chem., 35(3): 422-4/Marquet et al., 1996,Clin. Chem., 42(2): 258-62

-   -   CT: Motte et al., 1988, Clin. Chem. Acta, 174(1): 35-54/Niccoli        et al., 1997, J. Clin. Endocrinol. Metab., 82(2): 338-41    -   LDH: Meyers et al., 1992, J. Clin. Oncol., 10(1): 5-15

Other genetic markers can be used as, for example (De Vita, Hellman &Rosemberg, “Cancer, Principles and Practice of Oncology”, 6^(th)edition, Edition Lippincott Williams & Wilkins, Chapter 26, page 653):

-   -   Breast Her2/NEU/ERBB2 amplification or BRCA1, BRCA2 mutation    -   Prostate: PSA Mrna    -   Bladder: TP53 mutation    -   Colon: KRAS mutation    -   Kidney: NF1, NF2 mutation

Each patent, patent application, publication and literaturearticle/report cited or indicated herein is hereby expresslyincorporated by reference.

While the invention has been described in terms of various specific andpreferred embodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

What is claimed is:
 1. A method of identifying an insulin-like growthfactor I receptor (IGF-IR) modulator comprising: a) contacting IGF-IRwith a humanized anti-IGF-IR antibody, wherein said anti-IGF-IR antibodycomprises a light chain and a heavy chain, said light chain comprisingthe CDRs of SEQ ID NO: 2, 4 and 6, and said heavy chain comprising SEQID NO: 8, 10 and 12; b) contacting the complex of (a) with a compoundlibrary; c) identifying a compound which disrupts the complex of (a);and d) determining whether the compound exhibits IGF-IR agonist orantagonist activity by measuring a native ligand induced cellproliferation, wherein the agonist or the antagonist activity indicatesidentification of an IGF-IR modulator.
 2. A method of identifying ahuman insulin/insulin-like growth factor I hybrid receptor (hybrid R)modulator comprising: a) contacting hybrid-R with a humanizedanti-hybrid-R antibody, wherein said anti-hybrid-R antibody comprises alight chain and a heavy chain, said light chain comprising the CDRs ofSEQ ID NO: 2, 4 and 6, and said heavy chain comprising SEQ ID NO: 8, 10and 12; b) contacting the complex of (a) with a compound library; c)identifying a compound which disrupts the complex of (a); and d)determining whether the compound exhibits hybrid-R agonist or antagonistactivity by measuring a native ligand induced cell proliferation,wherein the agonist or the antagonist activity indicates identificationof a hybrid-R modulator.
 3. A method of identifying an insulin-likegrowth factor I receptor (IGF-IR) and human insulin/insulin-like growthfactor I hybrid receptor (hybrid-R) modulator comprising: a) contactingIGF-IR and hybrid-R with a humanized anti-IGF-IR and hybrid-R antibody,wherein said anti-IGF-IR and hybrid-R antibody comprises a light chainand a heavy chain, said light chain comprising the CDRs of SEQ ID NO: 2,4 and 6, and said heavy chain comprising SEQ ID NO: 8, 10 and 12; b)contacting the complex of (a) with a compound library; c) identifying acompound which disrupts the complex of (a); and d) determining whetherthe compound exhibits IGF-IR and hybrid-R agonist or antagonist activityby measuring a native ligand induced cell proliferation, wherein theaqonist or the antagonist activity indicates identification of an IGF-IRand hybrid-R modulator.
 4. A method of identifying an insulin-likegrowth factor I receptor (IGF-IR) modulator comprising: a) screening alibrary of peptide sequences, wherein said library of peptide sequencesbind to IGF-IR, and wherein the library is derived from a peptidesequence comprising at least the 6 CDRs sequences of SEQ ID NOs 2, 4, 6,8, 10 and 12; and b) determining whether the amino acid sequenceisolated in (a) exhibits IGF-IR agonist or antagonist activity in celllines displaying IGF-IR, wherein the agonist or the antagonist activityindicates identification of an IGF-IR modulator.
 5. A method ofidentifying a human insulin/insulin-like growth factor I hybrid receptor(hybrid-R) modulator comprising: a) screening a library of peptidesequences, wherein said library of peptide sequences bind to hybrid-R,and wherein the library is derived from a peptide sequence comprising atleast one of the 6 CDRs sequences selected from the group consisting ofSEQ ID NOs 2, 4, 6, 8, 10 and 12; and b) determining whether the aminoacid sequence isolated in (a) exhibits hybrid-R agonist or antagonistactivity in cell lines displaying hybrid R, wherein the agonist or theantagonist activity indicates identification of an hybrid-R modulator.6. A method of identifying an insulin-like growth factor I receptor(IGF-IR) and human insulin/insulin-like growth factor I hybrid receptor(hybrid-R) hybrid-R modulator comprising: a) screening a library ofpeptide sequences, wherein said library of peptide sequences bind toIGF-IR and hybrid-R, and wherein the library is derived from a peptidesequence comprising at least the 6 CDRs sequences of SEQ ID NOs 2, 4, 6,8, 10 and 12; and b) determining whether the amino acid sequenceisolated in (a) exhibits IGF-IR and hybrid-R agonist or antagonistactivity in cell lines displaying IGF-IR and hybrid R, wherein theagonist or the antagonist activity indicates identification of an IGF-IRand hybrid-R modulator.
 7. The method of claim 1, wherein the nativeligand is selected from the group consisting of IGF1 and IGF2.
 8. Themethod of claim 2, wherein the native ligand is selected from the groupconsisting of IGF1, IGF2, and insulin.
 9. The method of claim 3, whereinthe native ligand is selected from IGF1, IGF2 and insulin.
 10. Themethod of claim 4, wherein the cell lines displaying IGF-IR are selectedfrom the group consisting of MCF-7, T47D, BT20, ZR-75-1, MDA-MB-231 forbreast cells, PC3 and DU145 for prostate cells, A549, A427 and SK-LU-1for lung cells, HT29, Colo205 and CaCo-2 for colon cells, BC-PAP, FROand ARO for thyroid cells, SK-OV-3 for ovarian cells, BxPC3, MiaPaCa-2and LN36 for pancreas cells, SK-ES-1 for renal adrenal cancer sarcomacells, Daoy, TE-671 and D283 Med for medulloblastoma cells, MM-1S andMM-1R for retinoblastoma multiple myeloma cells and SK-MEL-28 formelanoma cells.
 11. The method of claim 5, wherein the cell linesdisplaying hybrid-R are selected from the group consisting ofMDA-MB-231, MDA-MB-157, MDA-MB-468, MDA-MB-453 and ZR-75 for breastcells and BC-PAP for thyroid cells.
 12. The method of claim 6, whereinthe cell lines displaying IGF-IR and hybrid-R are selected from thegroup consisting of MCF-7, T47D, BT20, ZR-75-1, MDA-MB-231, MDA-MB-157,MDA-MB-468, MDA-MB-453 and ZR-75 for breast cells, PC3 and DU145 forprostate cells, A549, A427 and SK-LU-1 for lung cells, HT29, Colo205 andCaCo-2 for colon cells, BC-PAP, FRO and ARO for thyroid cells, SK-OV-3for ovarian cells, BxPC3, MiaPaCa-2 and LN36 for pancreas cells, SK-ES-1for renal adrenal cancer sarcoma cells, Daoy, TE-671 and D283 Med formedulloblastoma cells, MM-1S and MM-1R for retinoblastoma multiplemyeloma cells and SK-MEL-28 for melanoma cells.